Cryopreservative compositions and methods

ABSTRACT

This disclosure describes a cryopreservative composition and methods for storing cells. Generally, the cryopreservative composition includes a sugar component and a sugar alcohol component, and is effective for storing and recovering cells without requiring dimethyl sulfoxide (DMSO).

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation application of U.S. patent application Ser. No.15/381,530, pending, filed Dec. 16, 2016, which claims priority to U.S.Provisional Patent Application No. 62/268,155, filed Dec. 16, 2015, thedisclosures of which are incorporated herein by reference.

GOVERNMENT FUNDING

This invention was made with government support under EB016247 andEB023880 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

SUMMARY

This disclosure describes, in one aspect, a cryopreservative compositionfor storing cells. Generally, the cryopreservative composition includesa sugar component and a sugar alcohol component, and is effective forstoring and recovering cells without requiring dimethyl sulfoxide(DMSO).

In some embodiments, the sugar component can include trehalose,fructose, sucrose, glucose, or a combination of sugars.

In some embodiments, the sugar alcohol component can include sorbitol,ethylene glycol, inositol, xylitol, mannitol, or a combination of sugaralcohols.

In some embodiments, the cryopreservative composition can furtherinclude an additive such as, for example, a small molecule or acombination of small molecules. In some of these embodiments, theadditive can include an amino acid or a combination of amino acids. Incertain embodiments, the amino acid can include proline, valine,alanine, isoleucine, histidine, taurine, ectoine, betaine,dimethylglycine, ethylmethylglycine, or an RGD peptide.

In some embodiments, the cryopreservative composition can furtherinclude a cell. In some of these embodiments, the cell may be acryopreserved cell. In some of these embodiments, the cryopreserved cellcan be a viable recovered cryopreserved cell.

In another aspect, this disclosure describes a method of cryopreservinga cell. Generally, the method includes adding a cell to any embodimentof a cryopreservative composition a summarized above, freezing thecomposition, storing the frozen composition at a temperature below 0°C., thawing the composition, removing the cell from the thawedcomposition, and culturing the cell under conditions effective for thecell to remain viable.

In some embodiments, freezing the composition can include at least oneround of cooling, re-warming, and further cooling.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. Differential Evolution (DE) Algorithm flow chart with blackboxes representing DE algorithm steps, and red boxes representingexperimental steps. The DE algorithm produces a population in gen 0 thatrandomly spans the parameter space, and a trial population (gen 1) thatis based on mutation of gen 0. These are both experimentally tested bythe user and the results are input into the DE algorithm, producing anemergent population which is further mutated and iterated in subsequentexperiments. As the algorithm converges, an optimum solution can beidentified.

FIG. 2. Trehalose, glycerol, ectoine, 1° C./min DE algorithm results forJurkat cells. (A) Cumulative best member solution. Recovery associatedwith the best solution increases and plateaus as the algorithmconverges. (B) Number of improved solutions per generation. The numberof improved solutions in each generation decreases and reaches zero whenthe algorithm has converged. (C) Emergent population with thegenerational average overlaid. The emergent population improves andeventually stops changing as the DE algorithm converges. This isreflected in the generational average, which increases and begins toplateau as the algorithm converges. The optimum composition identifiedby this run of the algorithm was 150 mM trehalose, 10% glycerol, 0.1%ectoine for Jurkat cells frozen at 1° C./min.

FIG. 3. Trehalose, glycerol, ectoine, cooling-rate DE algorithm resultsfor Jurkat cells. (A) Cumulative best member solution, this increasesand plateaus as the DE algorithm converges. (B) Number of improvedsolutions per generation, this decreases until it reaches zero as the DEalgorithm converges. (C) Emergent population with the generationalaverage overlaid. Pie charts at each average show the cooling ratedistribution within each generation. The optimum composition identifiedby this run of the DE algorithm was 300 mM trehalose, 10% glycerol,0.01% ectoine at 10° C./min cooling.

FIG. 4. High throughput concentration study confirmation of Jurkat DEalgorithm results. Colors corresponding to recovery values are plottedin squares corresponding to solution compositions within the algorithmparameter space. This concentration study performed at 10° C./minidentified the solution composition associated with maximum recovery tobe: 300 mM trehalose, 10% glycerol, and 0.01% ectoine (corresponds tothe points 5-Trehalose, 5-Glycerol, 1-Ectoine in the heat map above).This composition is the same as the composition identified by the DEalgorithm, confirming that it is indeed the optimum within the parameterspace at 10° C./min.

FIG. 5. Sucrose, ethylene glycol, alanine, taurine, ectoine,cooling-rate algorithm results for MSCs. (A) Cumulative best membersolution, this increases and plateaus as the DE algorithm converges. (B)Number of improved solutions per generation, this decreases until itreaches zero as the DE algorithm converges. The optimum compositionidentified by this run of the DE algorithm included: 300 mM ethyleneglycol, 1 mM taurine, 1% ectoine with a 1° C./min cooling rate.

FIG. 6. Scale-up viability and recovery of Jurkat cells (A,B) andmesenchymal stem cells (C,D) frozen in DE algorithm optimized solutions.Each cell type was frozen at 10° C./min in a TGE solution optimized forJurkats (TGE 10° C.), and a SEGA solution optimized for MSCs (MSC 10°C.). Results were compared to cells frozen in DMSO at 1° C./min (DMSO 1°C.) as it represents the current gold standard for both cell types.Jurkats performed well in both the SEGA 10° C. and TGE 10° C. solutions,and Jurkat viability was significantly higher in TGE 10° C. than bothSEGA 10° C. and the DMSO 1° C. control (A). MSCs also performed well inboth SEGA 10° C. and TGE 10° C. solutions, and had significantly higherrecovery in the SEGA 10° C. solution than both TGE 10° C. and the DMSO1° C. control (D). Significance markers indicate p<0.05.

FIG. 7. Osmolarity tests of individual molecules versus solutionsdesigned for storage of MSCs. (A) Mesenchymal stem cells were combinedwith: a full solution with sucrose, ethylene glycol, alanine, taurine,and ectoine (first column from left); individual components at the sameconcentration of each component as is present in the full solution(columns 2-6 from the left); individual components at concentrationscalculated to produce an osmolarity equal to the full solutionosmolarity (columns 7-11 from the left); or a10% DMSO positive controlsolution (right column). These results show that the full solutionoutperformed each individual component both at the concentration thatthe component is present in the full solution, and full osmolarityconcentrations, indicating that the full solution is stabilizing thecells in some way other than by osmolarity alone. (B) Osmolarities (mM)of the solutions tested in (A).

FIG. 8. Sugar synergy study. Jurkat cells were combined with solutionsof sugars at the indicated concentrations (150 mM total, 300 mM total,600 mM total). Solutions were composed of one sugar or equalconcentrations of two sugars, totaling the indicated concentration.Increasing the total sugar concentration increased recovery, butmultiple sugars at a given total concentration did not produce higherrecovery than a single sugar at that concentration. Disaccharides(trehalose, sucrose) outperformed monosaccharides (glucose, fructose).

FIG. 9. MSC vial recovery in MSC-designed and non-MSC-designedsolutions. MSCs were frozen at a rate of at −10° C./minute in a solutionof sucrose, ethylene glycol, alanine, taurine, and ectoine (SEGA) or asolution of trehalose, glycerol, and ectoine (TGE). The SEGA highsolution was designed for MSCs while the TGE high solution was designedfor Jurkats. The medium solution for each type produced <70% recoveryduring initial testing; the low solution for each type were produced<40% recovery during initial testing. (A) MSCs had higher viability inSEGA-high and TGE-high than medium and low solutions composed ofdifferent concentrations of the same components. (B) MSCs had higherrecovery in SEGA-high and TGE-high than medium and low solutionscomposed of different concentrations of the same components. MSCs frozenwith MSC-designed SEGA solutions tended to outperform their TGEcounterparts.

FIG. 10. Jurkat vial recovery in Jurkat-designed and non-Jurkat-designedsolutions. Jurkats were frozen at a rate of at −10° C./minute in asolution of trehalose, glycerol, and ectoine (TGE) or in a solution ofsucrose, ethylene glycol, alanine, taurine, and ectoine (SEGA). The TGEhigh solution was designed for Jurkats while the SEGA high solution wasdesigned for MSCs. The medium solution for each type produced <70%recovery during initial testing; the low solution for each type wereproduced <40% recovery during initial testing. (A) Jurkats had higherviability in TGE-high and SEGA-high than medium and low solutionscomposed of different concentrations of the same components. (B) Jurkatshad higher recovery in TGE-high and SEGA-high than medium and lowsolutions composed of different concentrations of the same components.Jurkat cells frozen with Jurkat-designed TGE solutions tended tooutperform their SEGA counterparts.

FIG. 11. Differential scanning calorimetry profile fortrehalose-containing solutions (T), glycerol-containing solutions (G),and ectoine-containing solutions (E). Samples were frozen to −100° C.(temperature range is plotted only to −30° C.), and freezing point andmelting point were reduced for solutions containing trehalose andglycerol. No glass transition was observed for any solution tested.

FIG. 12. Composite Raman images for penetrating and non-penetratingcryoprotectants. Glycerol was confirmed to be a penetratingcryoprotectant, as it is visible in both the cell space and the ice-voidspace. Trehalose, confirmed to be a non-penetrating cryoprotectant, isvisible in the void space only outside the cell, as evidenced by thedarker region in the trehalose image that corresponds to the area wherecell contrast is visible in the cell image.

FIG. 13. Cytochrome c distribution for cells frozen with trehalose,glycerol, and ectoine (TGE) solutions. The same TGE-high (good), med,and low (bad) solutions tested in FIG. 9 and FIG. 10 were combined withcells and imaged for cytochrome c using Raman microscopy. In primarilycells frozen with the good/high solution, cytochrome c is concentratedwithin mitochondria, a Raman marker that can be used to confirm a cellis alive. In primarily dead cells frozen with the low/bad solution,cytochrome c has been released and is disperse, a Raman marker that canbe used to confirm a cell is dead.

FIG. 14. Potential mean force distribution for lysozyme protein dimers.Potential mean force decreases as lysozyme proteins are moved closertogether in the presence of trehalose (blue), while potential mean forceincreases as they are moved closer together for the control. Thisindicates trehalose stabilizes proteins in low energy conformationsduring dehydration/freezing.

FIG. 15: Raman spectra of intracellular and extracellular compartmentsfor cells in a solution containing fructose. Arrows indicate peaksassociated with fructose.

FIG. 16. Cumulative best recovery rate for test solution (STG, SIM, XVM,MVG) and average recovery for DMSO at its optimum cooling rate.

FIG. 17. Number of improved solutions per generation for each solution(STG, SIM, XVM, MVG).

FIG. 18. Recovery reaches a maximum with appropriate incubation time forslow penetrating components (A) Fraction of live cells recovered forDMSO-free solutions SMC and SGC and DMSO solutions as a function ofincubation time prior to freezing at 3° C./min; (B) Raman imagesobtained of MSCs frozen at 3° C./min in SMC solution after 30 and 120minutes of incubation prior to freezing. Raman images are rendered forboth —CH and ice.

FIG. 19. Post thaw recovery of MSCs cryopreserved in SGC and SMC as afunction of total solution osmolarity. Linear best fit is given withcorrelation coefficient. (A) Recovery of cells has slight negativecorrelation for different osmolarity sucrose-mannitol-creatinesolutions. (B) Recovery of cells has slight positive correlation fordifferent osmolarity sucrose-glycerol-creatine solutions.

FIG. 20. Low temperature Raman microscopy of MSCs cryopreserved at 3°C./min in SGC with two different compositions. Images are rendered onice, osmolyte mixture and —CH. The fraction of cells with ice isdescribed for 10 cells measured.

FIG. 21. Algorithm iterations and post-thaw characterization ofoptimized solutions. (A) Algorithm iterations increase both attachmentand recovery of SGI solutions over five total generations. The analysisidentified solutions having recovery and attachment characteristicscomparable to DMSO frozen solutions. (B) Statistical replicates ofsolution iterations for SMC, SGC, and SGI tested in biologicaltriplicate. Immediate post-thaw recovery, and two hours post-thawattachment are reported for both experimentally and DMSO frozen cells.(C) DMSO recovery and attachment as a function of pre-freeze incubationtime. Longer pre-freeze incubation at room temperature in DMSO solutionssignificantly reduces attachment of cells, but has limited effect onrecovery.

FIG. 22. Post-thaw characterization of MSC differentiation,proliferation, and senescence. (A) Multi-lineage differentiationpotential is maintained in cells frozen in experimental solutions. Therewere no observable differences in chondrogenic and osteogenicdifferentiation and staining between experimental and fresh cells. (B)Proliferation rates were similar for SGI solutions compared to fresh andDMSO frozen cells (DMSO 0 hr) between two hours and 24 hours post-thaw.SMC and SGC solutions showed a slight reduction in proliferation ratefor the same time range. (C) Beta-galactosidase expression as anindicator of senescence was not markedly different between any fresh,DMSO or experimental samples tested, and each of these weresignificantly lower than positive control t-BHP induced samples.

FIG. 23. Gene expression profiles for H9-MSCs immediately post-thaw.This panel included genes for growth factors, adhesion molecules,transcription factors, chemokines, and stress genes. Each reported valuewas compared to a GAPDH internal control.

FIG. 24. Multicomponent solution effects on protein stability. (A) Ramanspectra of lysozyme in DPBS solution at room temperature and afterfreezing at −50° C. respectively. Spectra were normalized to the trpgroup. (B) Peak intensity ratio of α-helix at 1655 cm⁻¹ to Trp group at1553 cm⁻¹ for some of the solutions tested.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Certain medical conditions can be treated using cell therapies. Cellsused therapeutically are typically collected in one location, processedin a second location, and administered in a third location. The abilityto preserve cells enables coordination between manufacture of theproduct and patient treatment regimes. Maintaining the biologicalproperties of the cells during transit, processing, and/or storageincreases the likelihood that cell therapy employing the cells will beeffective.

Conventional methods of cryopreserving cells have involved the use ofdimethyl sulfoxide (DMSO). However, DMSO is systemically toxic in humansand can result in side effects ranging from mild (e.g., nausea andvomiting) to severe (e.g., cardiovascular and/or respiratorycomplications) when transfused in even trace amounts with thawed cells.Also, DMSO increases the mRNA level of the de novo DNA methyltransferaseDnmt3a accompanied by hypermethylation or hypomethylation of manygenetic loci. DMSO is thus unsuitable for use with reprogrammedtherapeutic cells such as, for example, induced pluripotent stem cellsor cells derived from them.

Certain cell types used therapeutically can exhibit altered/diminishedin vitro post thaw function. Mesenchymal stem cells can have diminishedindoleamine deoxygenase activity and/or altered cytoskeletal functionpost thaw. Natural killer cells used for immunotherapies also mayexhibit diminished post thaw function.

Moreover, survival for many cell types is strongly influenced by coolingrate, with a narrow range of cooling rates over which post thaw survivalis optimal. Freezing solution composition also influences cell survival,and changing the composition of the cryopreservation solution may changethe cooling rate at which optimum survival is observed. Cryopreservationprotocols are often determined empirically by changing composition andcooling rate until the desired outcome is obtained. This process istypically expensive, time consuming, and may not result in an optimizedprotocol.

A variety of strategies can be used to optimize processes with multipleinputs (e.g., composition of the freezing solution and cooling rate) andoutputs (e.g., recovery and viability). This disclosure describes theuse of the differential evolution (DE) algorithm (Storn, R. and Price,K., 1997, J Global Optim 11(4):341-359) to select composition andcooling rate for cryopreservation solutions. As described in more detailbelow, DE is applied for the purpose of determining DMSO-freecryopreservation solution formulations for different cell types (e.g.,lymphoblasts and mesenchymal stem cells). The methods involved have beenadapted to a high-throughput format: small numbers of cells are used anda small number of experiments are required. This type of approach cantransform the process of developing freezing protocols by reducing thenumber of cells and experiments required.

The first phase of this study involved using the DE algorithm to developa three-component cryopreservation solution used at a single coolingrate (1° C./min). Three components (trehalose, glycerol, and ectoine(TGE)) were selected as components of the exemplary freezing medium usedfor preservation of Jurkat cells, a hematopoietic model cell type, basedon pre-screening of multiple non-DMSO components. For this singlecooling rate study, the DE algorithm was programmed to output 18 vectorsolutions per generation with a weight=0.85 and a crossover=1. Jurkatcells cryopreserved in 10% DMSO at a cooling rate of 1° C./min were usedas a control.

For each generation of solutions tested, the scaled raw recovery of thebest solution increased or remained constant (FIG. 2A), while the numberof solutions that demonstrated improved recovery tended to decrease foreach generation (FIG. 2B). These results together (FIG. 2C) indicatethat the DE algorithm converged after six generations (e.g., sevenfreezing experiments) to an exemplary final solution composition of 150mM trehalose, 10% glycerol, and 0.1% ectoine (TGE, FIG. 2). The recoveryof Jurkat cells frozen in this exemplary TGE solution was 32%, almosttwice as high as the recovery of the control (16% recovery in 10% DMSOat 1° C./min).

Cooling rate influences cell survival, and the preferred cooling ratefor a given application can depend, at least in part, on the compositionof the freezing medium and the cell type being frozen. Therefore, theexemplary TGE solution composition identified for Jurkat cells at aconstant cooling rate of 1° C./min may not necessarily be the mosteffective composition at other cooling rates, and thus may not producethe highest recovery possible. To assess both cooling rate and thecomposition of the solution, the DE algorithm was programmed to output27 vector solutions per generation with weight=0.85 and a crossover=1.Solutions were separated into categories based on their DE algorithmdefined cooling rate, and were frozen in batches at a cooling rate of 0°C./min, 0.5° C./min, 1° C./min, 3° C./min, 5° C./min, or 10° C./min.Results were normalized and scaled raw recovery is reported, allowingresults from all cooling rates and all generations to be compareddirectly.

As with previous studies, the best member scaled raw recovery increasedor remained constant with increasing iterations (FIG. 3A) and the numberof improved solutions within each generation tended to decrease (FIG.3B). The number of solutions frozen at given cooling rates is describedby pie charts overlaid at the average recovery of each generation inFIG. 3C. These pie charts show that the DE algorithm quickly identifiespoor recovery in solutions frozen at 0° C./min (no freezing, recovery=0)and 0.5° C./min and eliminates these rates after 1-2 generations. Inearly generations, the majority of solutions with high recovery usedcooling rates of 5° C./min and 10° C./min. However, a spike in thenumber of 1° C./min solutions occurs in Generation 4 after the DEalgorithm identifies the same high recovery 1° C./min composition fromthe constant cooling rate study described in FIG. 2 above. Ultimately atconvergence, this DE algorithm run identified a TGE solution containing300 mM trehalose, 10% glycerol, 0.01% ectoine at a cooling rate of 10°C./min resulted in cell recovery for Jurkat cells of 35% recovery in theTGE solution at 10° C./min (compared to 16% recovery in 10% DMSO at 1°C./min). The DE algorithm converged after seven generations (or eightfreezing experiments).

High throughput screening of solution compositions was used to confirmthat the DE algorithm converged on the true optimum solution compositionfor a given cooling rate and component concentrations. Samples werefrozen and thawed at 10° C./min. Serial dilutions of glycerol, ectoinewere combined with dilutions of trehalose and cells suspended inNORMOSOL-R (Hospira, Inc., Lake Forest, Ill.) in 96-well plates. Thefinal concentrations in each well were equal to the full factorial arrayof concentrations used in the DE algorithm. The results from each wellwere normalized to a DMSO control included on each plate. Thisexperiment was repeated in triplicate and the recovery results from eachindividual composition were averaged and are plotted in FIG. 4 (standarddeviations were typically below 5%). This study confirmed that acomposition of 300 mM trehalose, 10% glycerol, and 0.01% ectoineresulted in the highest recovery for the array tested and confirmed theability of the DE algorithm to correctly identify an appropriate cellstorage solution for cooling at a rate of 10° C./min.

FIG. 2 and FIG. 3 relate to a three-component TGE solution tested withJurkat cells. To show that the DE algorithm is capable of converging tofreezing solution compositions using different solution components anddifferent cell types, a five-component combination of sucrose, ethyleneglycol, alanine, taurine, and ectoine (SEGA) was tested with mesenchymalstem cells at DE algorithm defined concentrations and cooling rates.These components were selected based on pre-screening experimentsperformed to identify combinations with high potential recovery. The DEalgorithm was programmed to output 27 vector solutions per generationwith a weight=0.85 and a crossover=1. Experimental testing and resultnormalization was similar to the methods described above.

As with previous experiments, the cumulative best member compositionincreased and the number of improved solutions decreased with eachgeneration. At convergence, this run of the DE algorithm identified aSEGA solution of 300 mM ethylene Glycol, 1 mM taurine, and 1% ectoineresulted in recovery for MSCs of 40% at 1° C./min (compared to 21%recovery in 10% DMSO at 1° C./min). Total convergence occurred afternine generations (10 freezing experiments) as evidenced by the increaseand plateau of the best member recovery (FIG. 5A) and the decrease inthe number of improved solutions per generation (FIG. 5B). Two of thecomponents tested (sucrose and alanine) were not present in the finalsolution formulation, indicating that the presence of these additivesdid not improve post thaw survival at this cooling rate.

Freezing experiments were performed in 1 mL vials to determine whetherthe results with low volumes and small cell numbers in 96-well studieswere reproducible when using larger, more clinically relevant volumes.DE algorithm solutions that resulted in maximum recovery were identifiedfor both Jurkats and MSCs at 10° C./min. These solutions are identifiedas TGE 10° C. (300 mM trehalose, 10% glycerol, and 0.01% ectoine at 10°C./min) and SEGA 10° C. (150 mM sucrose, 300 mM ethylene glycol, 30 mMalanine, 0.5 mM taurine, and 0.02% ectoine at 10° C./min). Thesesolutions were combined with cells, frozen, thawed, and analyzed forviability as described in Example 1. At least nine samples were analyzedfor each solution (batches of three or more on at least three differentdays). These were compared to solutions of cells in 10% DMSO frozen at1° C./min (the gold standard for both cell types, labeled DMSO 1° C.).

The TGE 10° C. solution resulted in significantly higher viability thanSEGA 10° C. and the DMSO 1° C. control (FIG. 6A) for Jurkat cells.Recovery was high across the board (>88%), but not statisticallysignificantly different for any of the solutions tested with Jurkatcells (FIG. 6B). Conversely, MSC viability testing showed nostatistically significant differences between solutions (FIG. 6C), whilethe SEGA 10° C. solution produced significantly higher recovery thaneither TGE 10° C. or DMSO 1° C. (FIG. 6D). This indicates that resultsfor an individual cell type are unique and can result in significantlyhigher viability (FIG. 6A) or recovery (FIG. 6D). However, both TGE andSEGA solutions produced acceptable viability and recovery in cell typesfor which they were not designed, indicating that DE-algorithm-designedsolutions may be used to successfully freeze multiple cell types.

FIG. 6 also shows that DE algorithm results are scalable. Although theimprovement in recovery for optimized solutions in comparison to DMSO issmaller in vial studies than in 96-well studies, this result may be due,at least in part, to the limited difference that is possible whenrecovery and viability are high. Cumulatively, these results support DEalgorithm testing of small volumes of cells and solutions to identifythose solutions that produce high viability and recovery at largervolumes.

Current methods of selecting a cryopreservation solution most often useempirical methods—e.g., testing a given composition and cooling rate andmeasuring post thaw recovery. This disclosure describes using a DEalgorithm to evaluate and identify cell storage solutions and/or coolingrates appropriate for a given type of cell. The DE algorithm can be usedfor different cell types and can concurrently evaluate both solutioncomposition and cooling rate. The DE algorithm as implemented rapidlyevaluated both solution composition and cooling rate using less than 200unique experimental points. Without the aid of the DE algorithm,approximately 7,000 unique experimental data points would have beenrequired to evaluate the compositions tested above. Best membersolutions increased and the number of improved solution compositionssteadily decreased with each advancing generation, consistent withconvergence of the DE algorithm. Convergence was confirmed byhigh-throughput studies using the same components and the same range ofconcentrations as the DE algorithm.

The ability to cryopreserve cells in a 96-well format enabled thetesting of generations with a large number of solutions (18-27).Freezing cells in 96-well format has been used to improve post thawrecovery of anchorage dependent cells. Cells cryopreserved in a 96-wellformat are also available commercially and used for drug screening andother applications. For example, conventional preservation of Jurkatscells and MSCs is typically performed using 10% DMSO and a cooling rateof 1° C./min. A limited number of studies have examined vitrification ofMSCs and the use of polymers to replace DMSO. This disclosure describesexemplary solutions with multiple components that preserve cellviability without DMSO. This disclosure describes, generally, thecombination of two or more cryoprotectants for effective cellcryoprotection. Additionally, these exemplary multi-componentcompositions result in cell viability and recovery higher than thatobserved using 10% DMSO, which is an important step forward towardsDMSO-free cryopreservation.

Exemplary cryoprotective solutions include those that may be used forstorage of lymphocytes. Different combinations of sugars, sugaralcohols, and amino acids were used as cryoprotective agents duringfreezing of lymphocytes using high throughput screening techniques.Statistical analysis of the data was performed using a Linear EffectsModel. The model associated with best fit was determined using BayesianInformation Criteria. A more detailed description of the analysis can befound in Example 2. 128 combinations of sugar, sugar alcohol, and aminoacid exhibited the highest predicted recovery rate. The ten bestperforming combination are given in Table 1.

TABLE 1 Combinations of additives that resulted in the highest post thawrecovery for lymphocytes cryopreserved in these combinations. SugarAlcohol Amino acid Trehalose Mannitol Ectoine Sucrose Sorbitol EctoineSucrose Sorbitol Taurine Sucrose Sorbitol Isoleucine Sucrose Sorbitol —Trehalose Mannitol — Trehalose Mannitol Valine Trehalose Sorbitol —Trehalose Xylitol — Trehalose Glycerol —

The data were further analyzed for highest recovery among thecombinations that include sucrose. Sucrose is an additive that isGenerally Regarded as Safe (GRAS). Thus, combinations that includesucrose may be of particular interest for regulatory and/orcommerciability of storage solutions for cryopreservation of cells forcell therapy applications. Other sugars may be equally suitable,however.

TABLE 2 Combinations with sucrose that result in high recovery. SugarAlcohol Amino acid Sucrose Glycerol Alanine, Creatine, Isoleucine,Taurine, or Valine Sucrose Arabitol Creatine, Isoleucine, or TaurineSucrose Mannitol Alanine, Creatine, Isoleucine, Taurine, Proline, orValine Sucrose Sorbitol Alanine, Creatine, Isoleucine, Taurine, Proline,or Valine Sucrose Xylitol Creatine, Isoleucine, Taurine, or ValineSucrose Glycerol Alanine, Creatine, Isoleucine, Taurine, or Valine

Table 2 lists multiple amino acids, each of which can be used incombination with the sugar and sugar alcohol. The listed amino acids maybe used alone or in combination with one or more amino acids.

Other exemplary compositions include those that may be used for storageof mesenchymal stem cells. Different combinations of sugars, sugaralcohols, and amino acids were used as cryoprotective agents duringfreezing of lymphocytes using high throughput screening techniques.Statistical analysis of the data was performed using a Linear EffectsModel. The model associated with best fit was determined using BayesianInformation Criteria. A more detailed description of the analysis can befound in Example 2. 76 combinations of sugar, sugar alcohol, and aminoacid exhibited the highest predicted recovery rate. The eight bestperforming combination are given in Table 3.

TABLE 3 Combinations of additives that resulted in the highest post thawrecovery for MSCs cryopreserved in these combinations. Sugar AlcoholAmino acid Sucrose Glycerol Isoleucine Sucrose Glycerol Ectoine SucroseGlycerol Alanine Sucrose Mannitol — Sucrose Mannitol Ectoine

The data were once again further analyzed for highest recovery among thecombinations that include sucrose. Sucrose is an additive that isGenerally Regarded as Safe (GRAS). Thus, combinations that includesucrose may be of particular interest for regulatory and/orcommerciability of storage solutions for cryopreservation of cells forcell therapy applications. Other sugars may be equally suitable,however. All sugar alcohols and additives listed in the combinations inTable 4 below are also GRAS.

TABLE 4 GRAS combinations with sucrose that result in high recoverySucrose Glycerol Alanine, Creatine, Isoleucine, Taurine Sucrose ArabitolCreatine Sucrose Mannitol Creatine, Taurine Sucrose Sorbitol Alanine,Creatine, Isoleucine, Taurine, Valine Sucrose Erythritol CreatineSucrose Inositol Creatine, Isoleucine

Table 2 lists multiple amino acids, each of which can be used incombination with the sugar and sugar alcohol. The listed amino acids maybe used alone or in combination.

In another evaluation, cells were combined with factorial combinationsof the components listed in Table 5, using one component from eachcategory. Plates including cells and solutions were frozen at coolingrates of 1° C./min, 3° C./min, 5° C./min, or 10° C./min.

TABLE 5 Components tested for both Jurkats (lymphocytes) and MSCs SugarsSugar Alcohols Additives (1-300 mM) (0.1-1.4M) (1-300 mM) SucroseArabitol Alanine Trehalose Erythritol Creatine Glycerol Ectoine InositolIsoleucine Mannitol Proline Ribitol Taurine Sorbitol Valine Xylitol

Recovery was calculated by dividing the number of live cells postthaw—calculated using a control curve correlating calcein-acetoxymethyl(AM) fluorescence to cell count—by the number of live cells seededpre-freeze (AO/PI counts taken prior to freezing). Recovery wasnormalized by dividing by the DMSO recovery on each plate, thenmultiplying by a standard DMSO recovery for each cooling rate. Thisnormalized recovery was used to compare samples between different platesand different cooling rates.

Statistical analysis of the data was performed using a Linear EffectsModel. The model associated with best fit was determined using BayesianInformation Criteria. 128 combinations of sugar, sugar alcohol, andamino acid were found to contain the highest predicted recovery rate forJurkats, while 76 combinations were found to contain highest predictedrecovery rate for MSCs. The ten best combinations for Jurkats aresummarized in Table 6 below, while the eight best combinations for MSCsare summarized in Table 7.

TABLE 6 Sugar Alcohol Amino acid Trehalose Mannitol Ectoine SucroseSorbitol Ectoine Sucrose Sorbitol Taurine Sucrose Sorbitol IsoleucineSucrose Sorbitol — Trehalose Mannitol — Trehalose Mannitol ValineTrehalose Sorbitol — Trehalose Xylitol — Trehalose Glycerol —

TABLE 7 Sugar Alcohol Amino acid Sucrose Glycerol Isoleucine SucroseGlycerol Ectoine Sucrose Glycerol Alanine Sucrose — Ectoine SucroseMannitol — Sucrose Mannitol Ectoine Trehalose — Ectoine

Statistical analysis revealed that the combination of sugar and coolingrate was most influential in predicting recovery. Jurkat cells performedbetter when frozen with sucrose at 10° C. and 1° C./min, or Trehalose at5° C./min. MSCs performed better when frozen with sucrose at 3° C. and5° C./min, or Trehalose at 10° C./min

Additionally, statistical analysis revealed positive and negativecorrelations with recovery for some of the compounds tested. In MSCs,recovery was positively correlated with glycerol and sorbitol. In Jurkatcells, recovery was negatively correlated with erythritol, ribitol, andproline (data not shown). In MSCs, recovery was negatively correlatedwith proline, valine, erythritol, and ribitol (data not shown).

To determine whether results of studies are due to interactions betweenmolecules or just due to the total concentration of cryoprotectants insolution, concentration studies were performed to compare the recoveryof cells in a full solution to cells in solutions of each individualcryoprotectant at both their concentration, and a concentration thatproduced the full solution osmolarity. Results for MSCs are shown inFIG. 7A.

FIG. 8 shows the results of a study to evaluate whether using multiplecomponents from the same category (e.g., multiple sugars or multiplesugar alcohols) would produce higher recovery. FIG. 8 shows thatincreasing the total sugar concentration increased recovery of Jurkatcells, but multiple sugars at a given total concentration did notproduce higher recovery than a single sugar at that concentration.Although recoveries are high for the 600 mM cases, this is anunrealistic sugar concentration for testing as it approaches thesolubility limits for sucrose and trehalose.

Sugar alcohol studies produced similar results, but a few combinationsof double sugar alcohols produced recoveries higher than eitherindividual sugar alcohol at the same concentration. Combinations withonly GRAS molecules that had favorable expected recovery based on thestatistics in ITDD experiments above are indicated in bold face in Table8.

TABLE 8 Sugar alcohol #1 Sugar alcohol #2 Arabitol Erythritol* GlycerolInositol Sorbitol Erythritol* Inositol Xylitol Glycerol InositolMannitol Sorbitol Xylitol Ribitol* Xylitol Sorbitol Xylitol *sugaralcohols that negatively influenced Jurkat cell recovery

Vial experiments were performed in order to ascertain if the recoverytrends observed at the 96-well scale were translatable to largerclinical volumes. Jurkat cells and MSCs were suspended in NORMOSOL-R(Hospira, Inc., Lake Forest, Ill.) and combined stepwise with 2×concentrations of optimized cryopreservation solutions. In addition torunning the best cryopreservation solutions, a medium-performingsolution and a poor-performing solution containing the same componentsat different concentrations were also tested. SEGA (sucrose, ethyleneglycol, alanine, taurine, ectoine) solutions were tailored for MSCs,while TGE (trehalose, glycerol, ectoine) solutions were tailored forJurkat cells. MSC experiments were run over at least three differentdays, with 3-6 vials of each solution in each run. Jurkat cellexperiments need higher n; data in FIG. 9 is reflective of a minimum ofone run (3 vials) on one day for some of the Jurkat cell samples.

SEGA high recovery was significantly greater than all other experimentalsolutions, and not significantly different than DMSO for MSCs. Similarexperiments were performed with Jurkat cells.

FIG. 10 shows that solutions tailored for Jurkats (TGE) outperformedsolutions optimized for MSCS (SEGA). The solution tailored for use withJurkat-cell-designed solution (TGE good, the TGE high bar in FIG. 10)outperformed all others in both viability and recovery. (FIGS. 10A and10B).

FIG. 16 and FIG. 17 show data for alternative solutions tailored for usewith Jurkat cells. Of the solution tested in FIG. 16 (Example 3), thesolutions with the highest rate of recovery were the STG (sucrose,sorbitol, taurine, glycerol) and MVG (sucrose, mannitol, valine,glycerol) solutions. The SIM (sucrose, sorbitol, isoleucine, mannitol)and XVM (sucrose, xylitol, valine, mannitol) solutions had lowerrecovery rates that were still better than DMSO, however. The highestrecovery rate at the end of each generation for each solution is shownin FIG. 16. XVM solutions trials were discontinued after generationfive. Thus, six generations were tested for XVM, and seven were testedfor STG, SIM, and MVG. In general, the highest recovery continuedimproving for each generation, and plateaued for all compositions exceptSTG.

The number of compositions with improved recovery rates for eachgeneration was counted for each solution and shown in FIG. 17. Eachsolution composition had a decreasing trend in the number of improvedsolutions as generation advanced.

Each of the STG solutions included sucrose, glycerol, sorbitol andtaurine, whereas the MVG solutions included sucrose, glycerol, mannitol,and valine. Without wishing to be bound by any particular theory, onepossibility is that different components of the composition could affectdifferent proteins or different areas of the cell. One example of cellstructure that could be affected differently by different osmolytes isthe cell membrane. The cell membrane can be irreversibly damaged byvolume changes and/or ice formation during cryopreservation, which canresult in cell lysis. Both surface and internal membranes are potentialareas of membrane cell injury regardless of cooling rate. Normal cellshave a layer of water surrounding the cell surface, which helps maintainprotein folding. During freezing, however, the concentration of liquidwater decreases, which can lead to destabilization of proteins in thecell membrane. In the solutions used in this study, each component coulddiffer in factors such as, for example, location (intracellular orextracellular), the macromolecules it stabilizes, and/or the surfacearea it stabilizes. The differences in the components could help withprotecting multiple cellular structures, thus decreasing the cumulativedamage to the cell.

For example, sugars can provide stabilization by replacing watersurrounding membranes during dehydration. Larger sugars can providebetter protection because they can insert between the phospholipid headsand create space for additional binding. Binding of sugars to membranescan increase the rigidity of the membrane, which can provide greaterresistance to disruption. Cells can dehydrate at slow cooling rates.During thawing, changes in the protein environment can inducedenaturing, but osmolytes including, for example, sugars can help withstabilization. This can reduce damage to membrane proteins and internalproteins.

Sugars are hydrophilic and cannot cross the plasma membrane withoutassistance from transporters. Disaccharides are more likely to belocated outside the cell and, therefore, are in a better position tostabilize the outer membrane. Additionally, disaccharides can bind moreof the membrane than monosaccharides because of their size.

Next, samples of selected experimental solutions tested (no cells) wereplaced in sample pans and run down to −100° C. (only graphed to −30° C.in FIG. 11) using the same freezing protocol as used with cells in thecontrolled rate freezer. The freezing and warming profiles for thesamples are shown in FIG. 11. The melting point and freezing point arereduced when there are more components in solution, which is an effectof increased concentration—higher osmolarity of the solution lowers thefreezing point and melting point. However, osmolarity alone is notenough to affect cell recovery. A solution of PBS, which is roughly 300mOsm does not result in high cell recovery. Also, there is no change insolution behavior at these solution concentrations below −30° C. Thereis no eutectic peak indicating that a glass transition occurred in anyof the samples at these concentrations (not shown), and one would expectto see glass transition at around −80° C. if it existed. Because thisphysical change is absent, the mechanism of protection for thesesolutions may be biological.

Raman microscopy data provides an indication of the location of actionof each cryopreservative. The images in FIG. 12 show Jurkat cells frozenin either glycerol (penetrating) or trehalose (non-penetrating). Eachcontrast photo shows brighter colors (yellow and white) to indicatesomething is present, and darker colors (red and black) to indicatesomething is at lower concentrations or absent. Cell contrast shows thatthe cells have rounded morphology, and ice contrast shows the voidbetween ice crystals, which includes both a cell and super cooledsolution that has not crystalized. The glycerol contrast image showsthat glycerol is present in the entire ice contrast void space,indicating that it is present both inside the cell and in the supercooled liquid surrounding the cell. The trehalose contrast image showsthat trehalose is excluded from the cell and is only present in thesuper cooled liquid surrounding the cell. This suggests that trehalosedoes not penetrate the cell even at freezing temperatures and, thus,acts on the cell membrane.

These experiments were performed for most of the components described inthe sections above both at room temperature and at freezing temperaturesto assess whether penetration was temperature dependent. The results ofthese penetration studies are summarized in Table 9.

TABLE 9 Category Component Penetration Sugar Trehalose Semi* FructoseYes Sucrose Yes Glucose Yes Sugar alcohols Glycerol Yes Sorbitol YesEthylene glycol Yes Inositol Yes Xylitol Yes Mannitol Yes Amino AcidsProline Yes Alanine Yes Valine Yes Other additives Taurine Yes EctoineYes *A small fraction of trehalose penetrates

Molecules excluded from the cell even at freezing temperatures mustexert any biological protection on the cell membrane. Molecules that donot penetrate at room temperature and are found inside the cell atfreezing temperatures may be improving cryopreservation by enabling poreformation in the cell membrane.

This pore formation may be demonstrated to be non-lethal to cells bylooking at the distribution of cytochrome c within the cell: a dispersedistribution indicates that the cell is dead, while a concentratedpocket indicates that a cell is alive. An example of this distributionis shown in FIG. 13 for three example solutions, a good solution (300 mMtrehalose, 5% glycerol, 0.01% ectoine) that has high live cell recovery(>90%), a medium solution (6 mM trehalose, 10% glycerol, and 0.5%ectoine; <70% live cell recovery), and a poor solution (30 mM trehalose,0% glycerol, and 1% ectoine; <40% live cell recovery).

Molecular dynamics simulations measure potential mean force (PMF) inkcal/mol between two proteins in a one or two component solution at eachpoint of the simulation for 100 ns. The proteins are brought closertogether, which simulates dehydration. A reaction coordinate of 0indicates that the proteins are right next to each other, while areaction coordinate of 5 indicates that the proteins are as far apart aspossible within the simulation. As one decreases the reactioncoordinate, the PMF for proteins in trehalose decreases, indicating thearrangement is at an energy minimum for that system—i.e., is favorable.The opposite is true in the control. This means that adding trehalose tothe system helps stabilize proteins as they dehydrate.

FIG. 18-20 show analysis of alternative compositions containing eithersucrose, mannitol, and creatine (SMC) or sucrose, glycerol, and creatine(SGC). Incubation time influences cell responses to freezing, indicatingthat the cryoprotective solution require some time to permeate the celland/or otherwise exert their cryoprotective effect. An SMC solution andan SGC solution (Table 14, Example 4) were incubated with cells at roomtemperature in NORMOSOL-R (Hospira, Inc., Lake Forest, Ill.) for 0minutes, 30 minutes, one hour, two hours, or four hours beforeundergoing freezing at 3° C./min. Live cell recovery increased withincubation time and experienced a maximum at one hour for SGC samples,and two hours for SMC samples. (FIG. 18A).

MSCs were incubated for 30 minutes or 120 minutes in the SMCcomposition, frozen to −50° C., and imaged using low temperature Ramanspectroscopy. Cells incubated for 30 minutes exhibited large internalice crystals for 10/10 of the cells imaged. In contrast, cells incubatedfor 120 minutes exhibited ice in only 3/10 cells imaged. The formationof ice inside the cell is considered to be a damaging event. (FIG. 18B).

It is common for cryopreservation solutions to contain highconcentrations of cryoprotective agents and therefore exhibit highsolution osmolarity. For example, a 10% DMSO solution has an osmolarityof ˜1400 mOsm. MSCs suspended in different combinations of sucrose,mannitol, and creatine (SMC) or sucrose, glycerol, and creatine (SGC)were frozen at 3° C./min, thawed, and the post-thaw recovery wasmeasured. The post-thaw recovery of SGC and SMC were plotted as afunction of total osmolarity for a range of different testedcompositions (FIG. 19). For SMC solutions, the range of solutionosmolarities is low (<500 mOsm) and there is a weak negative correlationbetween osmolarity and post-thaw recovery of MSCs (FIG. 19(A)). Incontrast, SGC solutions were evaluated over a higher range ofosmolarities (<1200 mOsm) and exhibited a weak positive correlation withthe compositions tested (FIG. 19(B)). These weak correlations suggestthat higher solution concentration does not necessarily correlate tohigher levels of post-thaw recovery. Thus, osmolarity of thecryoprotective solution, alone, is not enough to predict cell recovery.

In order to understand differences in freezing response for differentcombinations of the same three osmolytes, freezing studies using twodifferent compositions of SGC (SGC-A and SGC-B, Table 14) were repeated(FIG. 20). MSCs were frozen under the same conditions (total osmolarityand 3° C./min cooling rate) and imaged using low temperature Ramanconfocal microscopy. Cells frozen in SGC-B exhibited ice inside thecells for 10/10 cells imaged. In contrast, cells cryopreserved in SGC-Aexhibited less intracellular ice formation (3/10 cells imaged), implyingthat 7/10 cells survived freezing. Post-thaw recovery trends(average±SEM, n=4) for cells frozen in SGC-A (0.82±0.07) and SGC-B(0.71±0.05) using conventional controlled rate freezing were consistentwith the ice formation trends observed using Raman confocalmicroscopy—i.e., SGC-A had higher recovery and fewer cells with icecrystal formation than SGC-B.

FIG. 21-23 show post-thaw function and epigenetic changes in mesenchymalstromal cells cryopreserved using multicomponent osmolyte solutions.Different combinations of sugars, sugar alcohols, and small moleculeadditives were tested to identify the concentrations of components insolution which, when combined with cells, resulted in maximum cellattachment at two hours post-thaw. FIG. 21A shows a representativegenerational progression for a solution of sucrose/glycerol/isoleucine(SGI). Over multiple generations, the post-thaw recovery of live cellsincreases, and the percentage of those recovered cells that are able toattach to a surface also increases. This iterative process identifiedfor three separate exemplary compositions that maximized cell attachmentusing different components in solution: SGC (0 mM sucrose, 1.25%glycerol, 2 mM creatine), SGI (30 mM sucrose, 5% glycerol, 7.5 mMisoleucine), and SMC (150 mM sucrose, 62.5 mM mannitol, 6.25 mMcreatine).

MSCs frozen in these formulations were tested for attachment andrecovery using statistical triplicates of biological replicates. FIG.21B shows that samples frozen in experimental solutions have differentattachment and recovery behavior. High performing combinations, such asSGI, display recovery and attachment that is not statistically different(p>0.05) from both fresh cells and samples frozen in DMSO without anyfurther incubation (FIGS. 21B and 21C, ‘DMSO 0 hr incubation’). Otherexperimental combinations including SGC and SMC displayed significantlylower recovery (p<0.05) compared to fresh samples, but had attachmentvalues that approached their total recovery.

Incubating the cells in DMSO prior to freezing did not alter cellrecovery, but significantly reduced the attachment of cells with adecreasing fraction of cells attaching with increasing time of DMSOexposure (FIG. 21C). Diminished cell attachment was observed for cellsincubated with DMSO that do not undergo freezing (FIG. 21B).

MSCs frozen in osmolyte-based freezing solutions retain characteristiccell surface markers, proliferation, and osteo-chondral differentiationpotential. Fresh MSCs, MSCs frozen in experimental solutions, andDMSO-frozen MSCs all showed normal expression of positive (>99% forCD73, CD90, CD105) and negative (<1% for CD45) surface markers. Thesesurface marker expression characteristics are well within conventionalthresholds for cell-surface phenotype expression, and show that freezingwith DMSO and with experimental solutions does not change the expressionof these markers significantly.

MSCs displayed normal multi-lineage differentiation in all samples, asshown in FIG. 22A. Micromass cultures treated with chondrogenic mediaall showed characteristic blue color after staining with Alcian blue,indicating that these cultures produced glycosaminoglycan (GAG) contentconsistent with chondrogenesis. Cell monolayers treated with osteogenicdifferentiation media showed characteristic red color after stainingwith Alizarin red, consistent with osteogenesis.

Analysis of proliferation (FIG. 22B) showed that proliferative capacitywas maintained in SGI samples and was similar to both fresh and DMSOfrozen samples, but was slightly reduced in SMC and SGC samples based onthe reduced slope of their growth curves between two hours and 24 hours.

Senescence (FIG. 22C) did not vary significantly between samples. Therewere slight differences in senescence per cell at two hours, and thesedifferences were reduced after 24 hours. All samples showedsignificantly lower senescence than positive control t-BHP treatedcells.

Certain genes within select gene categories were analyzed using qRT-PCR.H9-MSCs subjected to different freezing approaches were assayedimmediately post-thaw for the expression of genes related to trophicfactor secretion such as HGF, VEGF, FGF2, CXCL12 (SDF-1a), mesodermallineage markers TWIST1, TWIST2 (DERMO1), MSX2, the anti-apoptotic markerBCL2, surface markers for cell adhesion such as CD106 and CD54, theosmotic regulator marker GAL-1, and stress-response markers such as EGR1and NFE2L2 (NRF2). The gene expression data for these genes issummarized in FIG. 23. These data show that the levels of HGF, ananti-scarring and anti-apoptotic factor, showed no differences betweendifferent freezing treatments, while fresh, non-frozen cells showed thehighest level of HGF expression.

Different freezing conditions did not appear to have any marked effectsin the expression of VEGF, the mitogen FGF2, the mesodermal genes MSX2and TWIST1, the osmotic gene GAL or the anti-apoptotic gene BCL2.However, TWIST2 was elevated in almost all of the frozen groups (SMC,SGI, and DMSO freezing for one hour) except for SGC, which presentedTWIST2 transcript levels comparable to fresh samples. Another notablechange in gene expression was observed for CXCL12 in two treatmentconditions (frozen SMC and DMSO lhr). Expression of mRNAs for MSCsurface markers CD106/VCAM1 and CD54/ICAM1 was easily detected inH9-MSCs regardless of the anti-freeze solution. Assessment ofstress-response genes such as EGR1 and NFE2L2 showed while expression ofNFE2L2 did not differ among treatment groups, EGR1 levels were highlyexpressed in the Fresh Media lhr group and were significantly lower inthe Fresh lhr DMSO-incubated samples and in all frozen groups.

The cryopreservation compositions can stabilize cellular proteins.Differential scanning calorimetry was used to characterize denaturationtemperature T_(m) of lysozyme heating in single and multi-componentosmolyte solution (Table 16, Example 6, below). The denaturationtemperature of lysozyme increased in the presence of osmolytes. SSE-Bhad a slightly higher denaturation temperature than SSE-A, but thedenaturation temperature of lysozyme in solutions containing a singleosmolyte that is a component of the SSE compositions—i.e., sucrose,sorbitol and ectoine—is higher than the combination of the threeosmolytes.

Low temperature Raman spectroscopy was used to study the secondarystructure of lysozyme—as a model cellular protein—during freezing insingle and multicomponent osmolyte solutions. Raman scattering issensitive to changes in protein secondary structure, and the α-helixconfiguration of proteins can be detected. The spectra of lysozyme inDPBS at room temperature and −50° C. were normalized to the Raman peakat 1553 cm′ and are shown in FIG. 24A. The Raman peak at 1553 cm⁻¹originates from trp group (tryptophan residues) and this group isconsidered free from feature changes and relatively stable astemperature changes, so it is appropriate for normalization purposes.The peak intensity of α-helix at 1655 cm⁻¹ significantly decreased forlysozyme in DPBS after freezing.

The peak intensity ratio of α-helix at 1655 cm⁻¹ to the trp group at1553 cm⁻¹ was calculated and is shown in FIG. 24B. Lysozyme in DPBSbefore freezing and after freezing had the maximum and minimum value forthe ratio of α-helix/Trp respectively. For all other osmolytes, thisratio fell between the maximum and minimum value, suggesting osmolyteswere preventing the loss of lysozyme secondary structure to varyingdegrees. There were no significant differences in the secondarystructure of lysozyme frozen in SSE-A and SSE-B suggesting that bothsolutions were effective in stabilizing lysozyme.

The sugar component of the cryopreservation composition can include anysuitable monosaccharide or disaccharide. In some embodiments, the sugarcomponent can include a disaccharide such as trehalose, sucrose,lactose, or maltose. In some embodiments, the sugar component caninclude a monosaccharide such as, for example, glucose or fructose,galactose. Also, the sugar component can include any combination of twoor more sugars. In some embodiments, the composition can include adisaccharide. In certain embodiments, the sugar component can includetrehalose or sucrose.

Generally, and regardless of the particular cell type with which thecryopreservation composition is used, the sugar component of thecomposition may be provided at a minimum concentration of at least 1 mMsuch as, for example, at least 2 mM, at least 3 mM, at least 4 mM, atleast 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM,at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least50 mM, at least 100 mM, at least 150 mM, at least 200 mM, or at least250 mM. The sugar component may be provided at a maximum concentrationof no more than 500 mM such as, for example, no more than 475 mM, nomore than 450 mM, no more than 425 mM, no more than 400 mM, no more than375 mM, no more than 350 mM, no more than 325 mM, no more than 300 mM,no more than 275 mM, no more than 250 mM, no more than 225 mM, no morethan 200 mM, no more than 175 mM, no more than 150 mM, no more than 125mM, no more than 100 mM, no more than 90 mM, no more than 80 mM, no morethan 70 mM, no more than 60 mM, or no more than 50 mM. The sugarcomponent may be provided at a concentration within a range havingendpoints defined by any minimum concentration listed above and anymaximum concentration listed above that is greater than the minimumconcentration. When more than one sugar is present in the composition,the concentration of the sugar component reflects the totalconcentration of all sugars in the composition. Thus, in someembodiments, the sugar component may be present at a concentration offrom 0.1 mM to 300 mM such as, for example, from 100 mM to 300 mM.

The sugar alcohol component of the composition can include any suitablesugar alcohol. Exemplary suitable sugar alcohols include, for example,glycerol, sorbitol, ethylene glycol, inositol, xylitol, arabitol,erythritol, ribitol, or mannitol. In some embodiments, the sugar alcoholcomponent can include any combination of two or more sugar alcohols. Incertain embodiments, the sugar alcohol component can include glycerol,sorbitol, ethylene glycol, inositol, xylitol, or mannitol.

Generally, the sugar alcohol component of the composition may beprovided at a minimum concentration of at least 0.1 M such as, forexample, at least 0.2 M, at least 0.3 M, at least 0.4 M, at least 0.5 M,at least 0.6 M, at least 0.7 M, at least 0.8 M, at least 0.9 M, at least1.0 M, at least 1.1 M, at least 1.2 M, at least 1.3 M, or at least 1.4M. The sugar alcohol component may be provided at a maximumconcentration of no more than 2.0 M such as, for example, no more than1.9 M, no more than 1.8 M, no more than 1.7 M, no more than 1.6 M, nomore than 1.5 M, no more than 1.4 M, no more than 1.3 M, no more than1.0 M, no more than 0.90 M, no more than 0.8 M, no more than 0.7 M, nomore than 0.6 M, or no more than 0.5 M. The sugar alcohol component maybe provided at a concentration within a range having endpoints definedby any minimum concentration listed above and any maximum concentrationlisted above that is greater than the minimum concentration. When morethan one sugar alcohol is present in the composition, the concentrationof the sugar alcohol component reflects the total concentration of allsugar alcohols in the composition. Thus, in some embodiments, the sugaralcohol component may be present at a concentration of 0.1 M to 1.4 M.For example, certain embodiments can include glycerol at a concentrationof 0.6 M to 1.4 M. Other particular embodiments can include analternative sugar alcohol at a concentration of 0.1 M to 0.6 M.

Generally, the additive component can include an amino acid or othersmall molecule that contributes to the cryopreservation of cells withinthe solution. Exemplary small molecules are listed in Table 9.Additional small molecules that may be effective as an additivecomponent include, for example, betaine, isoleucine, valine,dimethylglycine, ethylmethylglycine, histidine, n-acetylcysteine, an RGDpeptide, or an antioxidant (e.g., a superoxide dismutase, glutathione,vitamin C, vitamin E, glutathione, lipoic acid, ubiquinol, uric acid,and/or alpha monothioglycerol).

The additive component of the composition may be provided at a minimumconcentration of at least 1 mM such as, for example, at least 2 mM, atleast 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM,at least 8 mM, at least 9 mM, at least 10 mM, at least 20 mM, at least30 mM, at least 40 mM, at least 50 mM, at least 100 mM, at least 150 mM,at least 200 mM, or at least 250 mM. The amino acid component may beprovided at a maximum concentration of no more than 300 mM such as, forexample, no more than 275 mM, no more than 250 mM, no more than 225 mM,no more than 200 mM, no more than 175 mM, no more than 150 mM, no morethan 125 mM, no more than 100 mM, no more than 90 mM, no more than 80mM, no more than 70 mM, no more than 60 mM, or no more than 50 mM. Theamino acid component may be provided at a concentration within a rangehaving endpoints defined by any minimum concentration listed above andany maximum concentration listed above that is greater than the minimumconcentration. When more than one amino acid is present in thecomposition, the concentration of the amino acid component reflects thetotal concentration of all amino acids in the composition. Thus, in someembodiments, the additive component may be present at a concentration of0.1 mM to 300 mM such as, for example, 3 mM to 150 mM.

Generally, the composition can be free of DMSO or at least substantiallyfree of DMSO. As used herein, “free of DMSO” refers to a compositionthat contains no more than trace amounts of DMSO and may be absolutelyfree of DMSO. As used herein, “at least substantially free of DMSO”refers to a solution that contains a level of DMSO that provides nogreater cryopreservation than the remaining components of thesolution—i.e., an amount of DMSO that is inconsequential to thefunctionality of the solution.

Thus, in one aspect, this disclosure describes a cryopreservativecomposition. Generally, the cryopreservative composition includes asugar component and a sugar alcohol component, as set forth in moredetail above. In some embodiments, the at least a portion of the sugarcomponent may not necessarily penetrate the cell membrane and,therefore, act on the out surface of the cell. In such cases, the sugarcomponent can include trehalose. In some embodiments, thecryopreservative composition can further include an additive component.Generally, the cryopreservative composition possesses an amount of DMSOthat provides no more cryoprotection than the remaining components ofthe composition without the DMSO.

In some cases, the cryopreservative composition further includes a cell.Initially, the cell may be added to the cryopreservative compositionprior to being cryopreserved and stored. In other cases, the cell may bebeing stored as a component of a frozen cryopreservative composition. Instill other cases, the cell may be a viable cell recoverable from athawed cryopreservative composition. As used herein, a “viable” cellincludes a cell that remains living—under culture conditions suitablefor the cell—after having been stored frozen in a cryoprotectivesolution, stored below 0° C., then thawed and removed from thecryoprotective composition.

Thus, this disclosure also describes a method of cryopreserving andstoring a cell. Generally, the method includes adding a cell to anyembodiment of the cryoprotective composition described above, freezingthe composition, storing the frozen composition at a temperature below0° C., thawing the composition, removing the cell from the thawedcomposition, and culturing the cell under conditions effective for thecell to remain viable.

In some embodiments, the method can include controlled rates of coolingand/or controlled rates of re-warming.

In certain embodiments the composition may be frozen using a protocolthat reduces the extent and/or likelihood that stochastic ice forms inthe cells as they are frozen within the cryoprotective composition. Insuch embodiments, freezing the cells can include one or more cycles ofcooling, re-warming, and re-cooling the cryoprotective composition towhich cells have been added. For example, the freezing protocol caninclude one or more cycles of cooling (at an exemplary rate of −50°C./minute) and re-warming (at an exemplary rate of +15° C./minute). Thusa complete freezing protocol can include, for example, cooling aspecimen (cells and cryopreservative composition) at a rate of −10° C.per minute until the temperature reaches 0° C., then holding thetemperature at 0° C. for 15 minutes. The specimen can then be cooled ata rate of −1° C. per minute until the specimen reaches a temperature of−8° C., followed by one or more cycles of more rapid cooling andre-warming. For example, the specimen may be cooled at a rate of −50° C.per minute until the specimen reaches a temperature of −45° C., thenwarmed at a rate of +15° C. per minute until the specimen reaches atemperature of −12° C., before being cooled to an appropriate storagetemperature (e.g., −100° C.). The final cooling step may involve coolingthe specimen at a rate of, for example, −0.5° C. per minute, −1° C. perminute, −3° C. per minute, −5° C. per minute, or −10° C. per minute,depending, at least in part, on the constituents of the cryopreservativecomposition and cells in the specimen.

In the preceding description and following claims, the term “and/or”means one or all of the listed elements or a combination of any two ormore of the listed elements; the terms “comprises,” “comprising,” andvariations thereof are to be construed as open ended—i.e., additionalelements or steps are optional and may or may not be present; unlessotherwise specified, “a,” “an,” “the,” and “at least one” are usedinterchangeably and mean one or more than one; and the recitations ofnumerical ranges by endpoints include all numbers subsumed within thatrange (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

In the preceding description, particular embodiments may be described inisolation for clarity. Unless otherwise expressly specified that thefeatures of a particular embodiment are incompatible with the featuresof another embodiment, certain embodiments can include a combination ofcompatible features described herein in connection with one or moreembodiments.

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1 Cell Culture

The lymphoblastoid cells used in this study (Jurkat cells, ATCC,TIB-1522) were cultured in medium composed of high glucose RPMI 1640(Life Technologies, Carlsbad, Calif.), and 10% FBS (Qualified, LifeTechnologies, Carlsbad, Calif.). Jurkat cells were cultured to maintaincell density within 1×10⁵ to 3×10⁶ cells/mL. The mesenchymal stem cells(MSCs) used in this study were derived from H9 embryonic stem cells aspreviously described (Trevedi et al., 2008, Exp Hematol 36:350-359). MSCmedium was composed of aMEM base with glutamine (powder, LifeTechnologies, Carlsbad, Calif.), 10% FBS (Qualified, Life Technologies,Carlsbad, Calif.), and 1% non-essential amino acids (Life Technologies,Carlsbad, Calif.). Tissue culture flasks were coated with 0.1% porcinegelatin (Sigma-Aldrich, St. Louis, Mo.) for a minimum of two hoursbefore cell seeding. MSCs were seeded in gelatin-coated flasks at adensity of approximately 2500 cells/cm′. Cells were split or used forexperiments when they reached approximately 70-80% confluence and wereused for experiments only from passages 8 to 12.

Algorithm

The DE algorithm used in this study was developed from strategy 2(DE/local-to-best/1, which balances robustness and convergence) by Stornand Price (1997, J Global Optim 11(4):341-359) and was coded in MATLABwith discrete parameter values. The DE algorithm utilizes stochasticdirect search and independently perturbs population vectors to identifya global maximum within the user-defined parameter space. Briefly, theDE algorithm randomly generates an initial population (generation 0)that spans the entire parameter space. This population is composed of agiven number of solutions expressed as vectors (a set of numbers), andthe number of different solution components being tested defines howmany slots the population vectors have. Experimentally, these vectorscorrespond either to the different levels of solute in a solution or todifferent cooling rates (Table 10). Cells are frozen at DE algorithmdictated cooling rates with solutions made from these vectorspecifications, and the resulting experimental recoveries are iteratedback into the DE algorithm.

The DE algorithm utilizes this experimental information to modify theexisting population vectors, and predicts solutions that may result inmore favorable recovery. Briefly, the algorithm mutates existing vectorsto generate new test vectors, and performs head to head comparisons ofthe resulting experimental recovery of each of the population slots. Thebest value from this comparison (either the original or the new mutantvector) is stored in an emergent population. This mutation/comparisonprocess is repeated for all subsequent generations (FIG. 1) and resultsin an emergent population that changes less and less as the algorithmconverges. The final emergent population contains a set of solutionsthat have all been independently optimized using stochastic directsearch and contains the best possible compositions for freezing cellswithin the defined parameter space. Convergence can be measured byobserving an increase in cumulative best member recovery, a decrease inthe number of improved solutions within the emergent population aftereach generation, or by the generational average, which captures boththese metrics.

For these experiments, the generation size was set to either 18 or 27,the crossover set to 1, and the weight set to 0.85. The concentration ofeach component was allowed to vary discretely between 0 and the maximumsthat were identified from the literature or dictated by solubilitylimits. The concentrations used for each discrete level are listed inTable 10.

TABLE 10 Concentration levels and cooling rates used for each componentin the DE algorithm Level Level 1 Level 2 Level 3 Level 4 LevelComponent 0 (0) (1/100) (1/50) (1/10) (1/2) 5 (1) Trehalose 0 3 mM 6 mM30 mM 150 mM 300 mM Glycerol 0 13.7 mM 27.4 mM 137 mM 685 mM 1370 mM(v/v %) 0.1%  0.2%   1%   5% 10% Ectoine 0 0.7 mM 1.4 mM 7 mM 35 mM 70mM (w/v %) 0.01% 0.02% 0.1% 0.5%  1% Sucrose 0 3 mM 6 mM 30 mM 150 mM300 mM Ethylene 0 3 mM 6 mM 30 mM 150 mM 300 mM glycol Alanine 0 3 mM 6mM 30 mM 150 mM 300 mM Taurine 0 0.5 mM 1 mM 5 mM 25 mM 50 mM Coolingrate 0 0.5° C./min 1° C./min 3° C./min 5° C./min 10° C./min (differentscaling)

High Throughput 96-Well Plate Freezing

In both screening and DE algorithm experiments, cells were frozen in96-well plates to limit the number of cells and volumes of reagentnecessary, and to increase the number of samples that could be tested atone time. Solutions were made at 2× their final concentration indistilled H₂O (diH₂O). Cells were concentrated in NORMOSOL-R (Hospira,Inc., Lake Forest, Ill.) and combined 1:1 with 2× solutions in clearbottom black 96-well plates (Corning, Corning, N.Y.) to produce a 1×concentration of cryoprotectant solution (total volume 50 μL). As acontrol, wells of 10% DMSO solution were also included on each plate tonormalize results between all experiments. All samples were run intriplicate wells on each plate. Plates were sealed with molded siliconeround well covers (Laboratory Supply Distributers, Millville, N.J.) toprevent desiccation during freezing and storage. Plates were placed in arack in a controlled rate freezer, and frozen using the profile below:

1. Starting temp 20° C.

2. −10° C. per minute to 0° C.

3. Hold at 0° C. for 15 min

4. −1° C./min to −8° C.

5. −50° C./min to −45° C.

6. +15° C./min to −12° C.

7. −0.5, −1, −3, −5, or −10° C./min to −100° C. (as dictated by the DEalgorithm)

The rapid cooling and re-warming in steps 5 and 6 are included topromote ice crystal nucleation outside the cell before slow coolingproceeds, discouraging stochastic ice formation within cells.

Vial Freezing

Freezing of cells in vials was performed to compare the post thawrecovery obtained in 96-well plates (50 μL) to that observed inconventional cryovials (1 ml). Briefly, solutions were prepared at 2×the final freezing concentration and added stepwise to cells inNORMOSOL-R (Hospira, Inc., Lake Forest, Ill.) at a 1:1 final volumeratio in a 1.8 mL Nalgene™ CryoTube vials (Nunc, Thermo Scientific,Waltham, Mass.). Vials were moved to the freezer immediately and werefrozen using the same protocol as 96-well plates.

Thawing

Both 96-well plate and vial samples were thawed using a 37° C. waterbath. Vials were submerged in a 37° C. bath (to just under cap level)and agitated until only a small ice crystal was present.

Plates were thawed using a 37° C. water bath as well. Briefly, plateswere submerged to half their height and agitated for 1 minute. At t=1minute, they were removed from the bath and the silicone cover wasremoved to observe samples as they thawed. The plates were returned tothe 37° C. water bath and submerged to half their height again. Whenopaque samples became transparent (approximately 1 minute after beingreturned to the water bath) the plates were removed for immediateaddition of viability dye. Thermocouple probe analysis of the freezingand thawing rate in different wells of a 96 well plate showed nosignificant difference existed in temperature profiles of the wellstested in experiments.

Viability Assessment

The viability of all cells was assessed before freezing usingfluorescent acridine orange/propidium iodide (AO/PI) using the methodpreviously described (Pollock et al., 2015, Cytotherapy 17(1):38-45).Briefly, AO/PI was added to cell samples, and live and dead cells wereenumerated using a hemocytometer (a minimum of 200 cells were counted).This method was also used to measure the viability and recovery of vialsamples immediately after thawing. Viability was determined by dividingthe number of live cells by the number of total cells. Recovery wasdetermined by dividing the number of live cells post thaw by the numberof live cells seeded pre-freeze.

After 96 well plates were thawed, calcein-acetoxymethyl (AM)/propidiumiodide was added to each well and the wells were covered from light andplaced in a 37° C. incubator for 30 minutes to allow the live cells tocleave calcein-AM. The plates were then analyzed for fluorescence on aplate reader. Raw fluorescence values were used to calculate the numberof live and dead cells present in each well by correlating to a controlcurve of unfrozen cells. The live cell recovery was calculated bydividing the number of live cells present in thawed samples (calcein-AMplate reader fluorescence) by the number of live cells seeded pre-freeze(AO/PI counts). To normalize results between different plates anddifferent cooling rates, these raw recoveries were divided by a controlwell containing 10% DMSO on the same plate, then multiplied by astandard DMSO recovery at each cooling rate to give the ‘scaled rawrecovery’.

Example Calculation

$\begin{matrix}{{\frac{{Raw}\mspace{14mu} {recovery}}{{DMSO}\mspace{14mu} {plate}\mspace{14mu} {recovery}}*{DMSO}\mspace{14mu} {standard}\mspace{14mu} {recovery}} = {{\frac{0.2}{015}*0.16} = {0.21 = {{scaled}\mspace{14mu} {raw}\mspace{14mu} {recovery}}}}} & \;\end{matrix}$

Statistics

Error bars represent standard deviations of a minimum of 9 samplemeasurements, taken from experiments performed in batches of 3-6 over aminimum of 3 different days. Statistical significance was determinedusing a students t-test, with a significance level of p=0.05.

Example 2 Cell Culture

A lymphocyte cell model (Jurkats, ATCC) was cultured in high glucoseRPMI 1640 (Gibco) in a 37° C. incubator at 5% CO₂. Cell concentrationswere kept between 1×10⁵ and 3×10⁶ cells/mL, and cells were used forexperiments or underwent media changes every 3-4 days.

Liquid-Handling Robot High Throughput Solution Screening

High throughput factorial screening experiments were performed on aBiomek Beckman FX 96 liquid handling robot. Briefly, 12.5 μl of an arrayof additives were transferred to nine 96-well plates containing 12.5 μlof different sugar alcohols. Cells (25 μl) diluted in either a sugar (ina solution of NORMOSOL-R; Hospira, Inc., Lake Forest, Ill.) or aNORMOSOL-R blank solution were transferred to these nine plates to givea final volume of 50 μl in each well. Cells suspended in 10% DMSO inmedia were also included as a row of each plate to serve asnormalization wells for plate-to-plate comparison.

When all plate volume transfers were complete, silicone plate coverswere used to seal each plate, and plates were transferred to acontrolled rate freezer (Kryo 10 Series III, Planer PLC, Middlesex,United Kingdom) and frozen at cooling rates ranging from 1° C./min to10° C./min. The concentrations of components present in solution arelisted in Table 11, and were selected based on values described inliterature or solubility limits. Component concentrations were 0 if notlisted in a solution.

TABLE 11 Solution component Concentration Sucrose 300 mM Trehalose 300mM Glycerol 300 mM Sorbitol 300 mM L-arabitol 300 mM Inositol  75 mMErythritol 100 mM Xylitol 400 mM Mannitol 250 mM Ribitol  75 mM Proline300 mM Alanine 300 mM Isoleucine  75 mM Creatine  25 mM Valine 100 mMTaurine  50 mM Ectoine  70 mM

Concentration

Combinations were iterated experimentally through a differentialevolution algorithm described in detail in Pollock et al.(Algorithm-driven optimization of cryopreservation protocols. Submittedto Tissue Eng Reg Med, October 2015). Briefly, the algorithm utilizesexperimental input to predict component concentrations that will resultin higher recovery. After several iterations, a solution composition canbe identified that maximizes cell recovery within the concentrationbounds provided.

Positive/Negative Solution Screening

In order to determine if all solution components identified by thealgorithm are necessary to produce maximal recovery, 96-well platefreezing studies were performed with and without components at theiralgorithm optimized concentrations. Additional studies with eachindividual component in solution at a concentration that realizes theoptimized solution osmolarity were also performed to determine whetherosmolarity or solution composition was the critical factor indetermining cell survival.

Scale Up Vial Freezing Studies

Vial freezes were performed to confirm that results from 96-well studieswere scalable to larger, more clinically relevant volumes. Briefly,cells suspended in NORMOSOL-R (Hospira, Inc., Lake Forest, Ill.) werecombined stepwise with an equal volume of 2× experimental freezingsolutions to give a final volume of 1 mL. Vials were capped and cooledin a controlled rate freezer using the same freezing protocols describedabove.

Thawing

Both vials and 96-well plates were thawed in a 37° C. water bath. Vialswere submerged halfway and agitated until only a miniscule ice pelletremained. Cells were assessed for viability immediately post thaw, andthe remaining volume was suspended in fresh pre-warmed media andcentrifuged. After pelleting, the cells were re-suspended in fresh mediaand cultured for 48 hours in a 37° C. incubator at 5% CO₂ to assesspost-thaw proliferation.

Plate thawing was performed by submerging 96-well plates halfway in a37° C. water bath. After 1 min, silicone plate covers were removed toobserve the thawing behavior of the wells. Plates were replaced in thewater bath for an additional 30 seconds to 1 minute until opaque frozensamples became transparent. Plates were then immediately processed forviability.

Viability and Functionality

Cells were assessed for viability at the conclusion of each experiment.Both pre-freeze counts, and all post-thaw vial counts (0 hr and 48 hr)were performed using acridine orange/propidium iodide (AO/PI). Briefly,cells were combined with AO/PI, loaded into a hemocytometer, and countedmanually using a fluorescent microscope. A minimum of 200 total cellswere counted for each sample.

Plate samples were also assessed for viability using fluorescence.Briefly, a solution of calcein-AM/propidium iodide was prepared and avolume of 50 μl was added to each sample well of experimental 96-wellplates, producing a 1:1 dilution. Plates were protected from light andplaced in an incubator for 30 minutes to allow for calcein-AM digestion.A fluorescent plate reader (Synergy HT, BioTek Instruments, Inc.,Winooski, Vt.) was used to determine the fluorescence in each well,which was correlated to live and dead cell counts using a control curve.

Viability was calculated for all samples by dividing the number of livecells by the number of total cells. Recovery was calculated directly bydividing the number of live cells post thaw by the number of live cellspre-freeze. Recovery for 96-well plate samples was further normalized;each experimental sample recovery was divided by the DMSO recovery onthe same plate, and then multiplied by the standard DMSO recovery forthe cooling rate of each experiment.

Differential Scanning Calorimetry

Differential scanning calorimetry was performed on a Perkin-Elmer Pyris1 according to the manufacturer's instructions. The solutions testedcontained trehalose, glycerol, and/or ectoine in the concentrationsshown in Table 12.

TABLE 12 Solution name Trehalose Glycerol Ectoine TGE 300 mM 5% 0.01% TG300 mM 5% 0 TE 300 mM 0 0.01% GE 0 5% 0.01% T 300 mM 0 0 G 0 5% 0.01% E0 0 0.01% Blank 0 0 0

Raman Microscopy

Confocal Raman Microspectroscopy (CRM) measurements are conducted usingWitec Confocal Raman Microscope System Alpha 300R (WITec,Wissenschaftliche Instrumente and Technologie GmbH, Ulm, Germany) withUHTS300 spectrometer and DV401 CCD detector. A wavelength of 532 nmNd:YAG laser and 10 mW of power is used as excitation source. The laseris transferred to the microscopy in a singer fiber. A 100× air objective(NA 0.90; Nikon Instruments, Melville, N.Y.) is used for focusing the532 nm excitation laser to the sample. Data collection and analysis areperformed by Windows-based WitecControl_1.38 software.

Molecular Dynamics Simulations

Gromacs molecular dynamics software (Berendsen et al., 1995, Comp PhysComm 91:43-56; Páll et al., 2015, Proc EASC 2015 LNCS 8759:3-27) withthe Martini coarse grain force field was used to perform moleculardynamics simulations. A lysozyme dimer (3VFX from the protein data bank)was placed in a first solution containing coarse-grain water and also ina second solution containing water and trehalose. The dimer was orientedwith respect to the x-axis. A pulling simulation was conducted, movingthe two lysozymes apart from their initial positions to a separation of5 nm between their centers of mass. From this pulling simulation, thepositions at every distance were taken and an individual moleculardynamics simulation was conducted at each distance using a potential(spring) to fix the monomers at the specified distance. From thesimulation outputs at every distance, a weighted histogram analysismethod was used to construct a free energy curve.

Statistics

To assess the importance of the four explanatory variables of interest(Sugar, Rate, Additive and Alcohol), without neglecting the variationbetween each batch and between each plate within the batches, we soughtto find the best Linear Mixed Effects (LME) model. With y_(i) the i'thoutcome of the response, X_(i) the corresponding values of thecovariates, the LME model is defined as

y _(i) =X _(i) β+Z _(i)γ+ε_(i),

where β is a vector with the fixed effects, γ is a vector with randomeffects normally distributed with mean 0, and Z_(i) is the design matrixfor the random effects. ε_(i) is the error term, normally distributedwith mean 0.

To select the most fitting model, we performed backward eliminationbased on the Bayesian Information Criterion (BIC) starting with a modelthat includes all variables and second order interactions. Furthermore,we included two random variables, one to catch the effect of the batch,and one to catch the effect of each plate within the batch—i.e., thefull model is:

y _(i) =x _(i,Sugar)β_(Sugar) +x _(i,Rate)β_(Rate) +x _(i,Alcohol) +x_(i,Additive)βAdditive+x _(i,Sugar×Rate)β_(Sugar×Rate) +x_(i,Sugar×Additive)β_(Sugar×Additive) +x_(i,Sugar×Alcohol)β_(Sugar×Alcohol) +x_(i,Rate×Additive)β_(Rate×Additive) +x _(i,Rate×Alcohol)β_(Rate×Alcohol)+x _(i,Additive×Alcohol)β_(Additive×Alcohol) +z _(i,Batch)γ_(batch) +z_(i,Plate)γ_(Plate)+ε_(i),

where the x's are the covariates, β's are the corresponding fixedeffects, z's are covariates for the random effects,γ_(Batch)˜N(0,σ_(Batch)) is the random effect for the Batch variable,and γ_(Plate)˜N(0, σ_(Plate)) is the random effect for the Platevariable. Finally, ε_(i)˜N (0, σ_(ε)) is the error term.

The models were fitted using the lmer function from the R package lme4.The elimination was made using the step function from the lmerTest R.The only term dropped was the interaction between Rate and Alcohol.

Student's t-tests were also performed to determine statisticallysignificant differences (p=0.05) between samples tested in thepositive/negative solution screening experiments and vial freezingexperiment sections described above.

Example 3

Lymphocytes (Jurkat cells) were prepared as described in Example 2. Nineexperimental solutions were tested in each generation of algorithmiteration, with concentrations ranging between 0 and the maximums listedfor each component in the solutions listed in Table 13 were testedagainst a solution of 10% DMSO. The cells were suspended in test wellsof a 96-well plate. Plates were covered and placed in the control ratefreezer. Cryopreservation was performed using a control rate freezer,set to cooling rates of 0.5° C./min, 1° C./min, 3° C./min, 5° C./min, or10° C./min. After freezing protocol, plates were stored in a deepfreezer until thawing.

TABLE 13 Solution Sugar Polyol Amino Acid name Sucrose Glycerol MannitolSorbitol Xylitol Ile Tau Val STG 300 mM 10% 300 mM 50 mM SIM 300 mM 250mM 150 mM 75 mM XVM 300 mM 250 mM 400 mM 100 mM MVG 300 mM 10% 250 mM100 mM

Thawing was performed in a 37° C. water bath. Plates were partiallysubmerged for one minute before removing the silicone lid covering eachwell. After lid removal, plates were partially submerged again untilsamples were just thawed (appearance changed from opaque to clear), andviability dye was added to each plate immediately.

Calcein-AM aliquots were suspended in 4 mL of 20 μg/mL propidium iodide(PI) solution to a final concentration of 2 μM calcein-AM and 20 μg/mLPI. A volume of 50 μL of the caleinAM/PI viability dye was added to eachwell in the plate. Plates were covered and placed in the incubator forthirty minutes, and then fluorescence was measured using a plate reader(BioTek Instruments, Inc., Winooski, Vt.) with ex/em florescence spectraof 485/528 (calcein-AM/live) and 530/590 (PI, dead). Live and deadfluorescence measurements were recorded for each well.

The average live and dead cell counts after thawing were calculated foreach solution for the test wells containing cells and control wellsloaded with NORMOSOL-R (Hospira, Inc., Lake Forest, Ill.) or DMSO bycomparing the in-well fluorescence to a control curve correlatingfluorescence and known cell count. Viability prior to freezing and cellsseeded per well were used to calculate the percentage of live cellsrecovered after thawing.

${{Recovery}(\%)} = {\frac{{Live}\mspace{14mu} {cells}\mspace{14mu} {post}\mspace{14mu} {thaw}}{{Live}\mspace{14mu} {cells}\mspace{14mu} {pre}\mspace{14mu} {freeze}} \times 100}$

The average live cell recovery, standard deviation and p-value comparedto the DMSO were calculated. The recovery was scaled to the average DMSOrecovery rate for each plate and normalized to the average DMSO recoveryvalue for each cooling rate. This correction allowed samples frozen ondifferent plates at different cooling rates to be compared.

Results are shown in FIG. 16 and FIG. 17.

Example 4 Cell Culture

Human H9 ESC derived mesenchymal stem cells (MSCs) were isolated aspreviously described (Trivedi et al., 2008. Exp Hematol 36(3):350-359).MSCs were cultured in alpha-MEM (Gibco, Thermo Fisher Scientific,Waltham, Mass.) supplemented with non-essential amino acids (NEAA,Gibco, Thermo Fisher Scientific, Waltham, Mass.) and 10% FBS (qualified,Gibco, Thermo Fisher Scientific, Waltham, Mass.) in a 37° C. incubatorat 5% CO₂. Cell confluency was maintained between 20% and 80% and mediawas changed every 3-4 days. Cells were used for experiments only betweenpassages 8-12.

Cell Freezing

Cells diluted in blank solution (NORMOSOL-R, Hospira, Inc., Lake Forest,Ill.) were transferred to freezing vials and an equal volume ofcryoprotectant solutions (Table 14)) at 2× their final concentrationswere added to the vials stepwise. 10% DMSO controls were also prepared,in which cells suspended in MSC media were added to vials and DMSOintroduced in the same stepwise fashion. For incubation studies, thesecell suspensions were incubated at room temperature for 0 minutes, 30minutes, one hour, two hours, or four hours before freezing according tothe following protocol using a controlled rate freezer (Kryo 10 SeriesIII, Planer PLC, Middlesex, United Kingdom):

1. Starting temp 20° C.

2. −10° C. per minute to 0° C.

3. Hold at 0° C. for 15 minutes

4. −1° C./min to −8° C.

5. −50° C./min to −45° C.

6. +15° C./min to −12° C.

7. −3° C./min to −100° C.

The final 1× concentrations present with cells for each solution testedare listed in the results section.

TABLE 14 Solution name Sucrose Mannitol Glycerol Creatine SMC 150 mM 125mM 12.5 mM SGC 150 mM 2.5% 12.5 mM SGC-A 150 mM 684 mM   25 mM SGC-B 300mM 684 mM 12.5 mM

Thawing

Frozen vials were thawed in a 37° C. water bath. Vials were submergedhalfway and agitated until only a miniscule ice pellet remained. Cellswere assessed for viability immediately post thaw.

Viability and Functionality

Cells were assessed for viability at the conclusion of each experiment.Both pre-freeze and post-thaw vial counts were performed using acridineorange/propidium iodide (AO/PI). Briefly, cells were combined withAO/PI, loaded into a hemocytometer, and counted manually using afluorescent microscope. A minimum of 200 total cells were counted foreach sample. Viability was calculated for all samples by dividing thenumber of live cells by the number of total cells. Recovery wascalculated directly by dividing the number of live cells post thaw bythe number of live cells pre-freeze.

Osmolarity

Osmolarity of solutions was measured using an osmometer (OSMETTE,Precision Systems Inc., Natick, Mass.) for each solution and allmeasurements were repeated in triplicate.

Differential Scanning Calorimetry Differential scanning calorimetry wasperformed on a differential scanning calorimeter

(Q1000, TA Instruments, New Castle, Del.). Experimental solutionswithout cells were frozen to −150° C. using the following protocol:

1. Set starting temperature to 20° C.

2. Cool to −150° C. at 10° C./min

3. Hold for 3 min at −150° C.

4. Warm to 20° C. at 10° C./min

Confocal Raman System

Confocal Raman Microspectroscopy (CRM) measurements were conducted usinga confocal Raman microscope system (Alpha 300 R, WITec WissenschaftlicheInstrumente and Technologie GmbH, Ulm, Germany) with a spectrometer(UHTS 300, WITec Wissenschaftliche Instrumente and Technologie GmbH,Ulm, Germany) and detector (DV401 CCD, Andor Technology Ltd., Belfast,United Kingdom) with 600/mm grating. The spectrometer was calibratedwith a Mercury-argon lamp. A wavelength of 532 nm Nd:YAG laser poweredat 10 mw was used as an excitation source. The laser was transmitted tothe microscopy in a singer fiber. A 100× air objective (NA 0.90; NikonInstruments, Melville, N.Y.) was used for focusing the 532-nm excitationlaser to the sample. Samples were frozen using a controlled temperaturestage as previously described (Dong et al., 2010. BiophysJ99(8):2453-2459).

Raman Measurement of Frozen MSC Cells

MSC cells were detached from the flask and washed with DPBS solutionbefore being suspended in experimental solutions. Roughly 1 μL of cellsuspension was placed on an aluminum sheet, covered with a piece of mica(Ted Pella, Inc., Redding, Calif.) and sealed with KAPTON tape (Dupont,Wilmington, Del.), to prevent evaporation/sublimation during eachexperiment. Cell suspensions were cooled to −6° C. at which point thesample was seeded by a nitrogen-cooled needle. Subsequently, thesolution was cooled down at 3° C./min to −50° C. Ten Raman images of 30μm×30 μm were collected.

Raman Image/Spectral Analysis

Spectrums at each pixel were analyzed using characteristic wavenumbersof common intracellular and extracellular materials (Table 15), and wereintegrated with background subtraction to result in an image. Spectrafor the osmolytes used in the investigation overlapped with each other,so a broad peak centered at 850 cm⁻¹ was used to generate Raman imagesfor all osmolytes. Data analysis was performed by Windows-based ProjectFOUR software plus version 4.0 (Microsoft Corp., Redmond, Wash.).

TABLE 15 Peak assignments for molecules of interest detected using Ramanspectroscopy. Component Frequency used for this study cm⁻¹ Ice 3120 (OHstretching) Amide I 1659 (C═O stretching) Glycerol  851 (C—C stretching)Sorbitol  878 (C—C═O stretching) Glucose  840 (C—C stretching) Sucrose 836 (C—C stretching) Creatine  840 (C—N torsion)

Statistics

Averages plus or minus standard error of the mean are reported unlessotherwise noted. Student's t-tests were performed to determinestatistically significant differences (p<0.05) between samples tested inthe osmolarity and sugar substitution studies.

Example 5 Cell Culture

The study involved the use of induced MSCs derived from H9 embryonicstem cells (H9-MSCs; Trivedi et al., 2008. Exp Hematol 36(3):350-359),which exhibit similar cell surface phenotype as bone marrow-derived MSCs(BM-MSCs), as well as appropriate in vivo migration and homing behaviorin mouse models. Media used with H9 MSCs was composed of aMEM base(Thermo Fisher Scientific, Waltham, Mass.), 10% FBS (qualified), and 1%non-essential amino acids (Thermo Fisher Scientific, Waltham, Mass.).Culture flasks were coated with 0.01% porcine gelatin (Thermo FisherScientific, Waltham, Mass.) for a minimum of 2 hours before H9 MSCseeding. H9 MSCs were seeded in gelatin-coated flasks at a density ofapproximately 2,500 cells/cm². Cells were split when they reached 70%confluence and were used for experiments only between passages 8 to 12.

Optimization

The differential evolution algorithm used in this study was developedfrom strategy 2 (DE/local-to-best/1, balances robustness andconvergence; Storn, R. & Price, K., 1997. J Global Optimization 11:341)and was coded in MATLAB. Details of the algorithm have been previouslydescribed (Pollock et al., 2016. J Tissue Eng Regen Med;doi:10.1002/term.2175). For this investigation, the algorithm was set toaccept and provide output for discrete parameter vectors. The weight wasset to 0.85, the crossover was set to 1, and cell attachment afterfreezing in each solution was used to iterate the cost function.

Surface Marker Characterization

Cells were suspended to a concentration of 1×10⁶ cells/ml in media andstained with a panel of the following antibodies: mouse IgG1 anti-humanCD73 (APC-conjugated, BD Biosciences, San Jose, Calif., clone AD2),mouse IgG1 anti-human CD90 (FITC-conjugate, clone 5E10, MolecularProbes, Eugene, Oreg.), mouse IgG1 anti-human CD105 (PE-conjugated, R&DSystems, Inc., Minneapolis, Minn. clone 166707) and mouse IgG1anti-human CD45 (BV421-conjugated, clone HI30, BD Biosciences, San Jose,Calif.). Cells were incubated with antibodies for 30 minutes at 4° C.Flow cytometry was performed on a flow cytometer (LSR II, BDBiosciences, San Jose, Calif.) at low flow rate with the fine adjustknob five turns from max. At least 15,000 events were recorded for eachsample. Cell populations were gated for forward and side scattercompared to unstained MSCs and CD45 expressing Jurkat cells as,respectively, negative and positive controls to establish boundaries forfluorescent signals.

Multilineage Differentiation

Differentiation of both fresh and post-thaw cells was induced using achondrogenesis kit (STEMPRO, Thermo Fisher Scientific, Inc., Waltham,Mass.) and osteogenesis media and protocols. Chondrogenic micromasscultures were stained with 1% Alcian blue solution, while osteogeniccultures were stained with 2% Alizarin red solution.

Vial Freezing

Optimized solutions were prepared at double (2×) the final freezingconcentration and added stepwise to cells in NORMOSOL-R (Hospira, Inc.,Lake Forest, Ill.) at a 1:1 final volume ratio in a freezing vial(NALGENE, Nalge Nunc, Penfield, N.Y.). Control cells in media weresimilarly combined stepwise with DMSO at a 1:1 final volume ratio. Eachof these vials was incubated at room temperature for 0 hours, one hour,or two hours. Experimental solutions were frozen using a 3° C./mincooling rate protocol described below, while DMSO solutions were frozenusing a 1° C./min cooling rate protocol on a controlled rate freezer(Kryo 10 Series III, Planer PLC, Middlesex, United Kingdom). Amulti-step procedure was followed in which the starting temperature wasset at 20° C., and temperatures were subsequently modulated as follows:−10° C./minute to 0° C., hold at 0° C. for 15 minutes, −1° C./min to −8°C., −50° C./min to −45° C., +15° C./min to −12° C., and finally −1° C.or −3° C./min to −100° C.

Thawing

Samples were submerged in a 37° C. bath to just under cap level, andagitated until only a small ice crystal was present. The cells werecombined with acridine orange/propidium iodide (AO/PI) and counted usinga hemocytometer. Samples were diluted and the supernatant was aspiratedafter centrifugation. Cells were then prepared for cellular assaysmeasuring proliferation, senescence or actin alignment, as well asbiochemical studies using isolated DNA and RNA.

Attachment and Proliferation

Cell attachment of samples post-thaw was measured using a fluorescentplate reader. Samples were re-suspended in media and seeded in each oftwo gelatin-coated, 6-well plates. After two hours or 24 hours, thesepaired plates were washed with PBS, 1 μM calcein-acetoxymethyl (AM) dyewas added, and then analyzed for fluorescence on a plate reader. Rawfluorescence values were used to calculate the number of live cellspresent in each well by correlating to a control curve of cells seededat known densities. The live cell attachment was calculated by dividingthe number of live cells present in the two-hour plated samples(calcein-AM plate reader fluorescence) by the number of live cellsseeded pre-freeze.

Senescence

Cell senescence of samples post-thaw was measured using a luminescentplate reader. Samples were re-suspended in media and equal parts wereadded to each of four gelatin-coated 6-well plates. After incubation topermit attachment, plates were washed with PBS and two plates wereanalyzed at a time, one for proliferation and one for senescence.Beta-glo (Promega, Madison, Wis.) luminescent dye was added to measuresenescence and 1 μM calcein-AM dye was added to measure proliferation.Plates were analyzed using a plate reader (BioTek Instruments, Inc.,Winooski, Vt.) for luminescence (Beta-glo plate) and fluorescence(485ex/528em, calcein-AM plate), respectively. A relative measure ofsenescence is reported here by dividing the base-corrected luminescence(approximation of total senescence) per well by the base-correctedfluorescence (approximation of total cells per well). H9-MSCs treatedfor one hour on seven consecutive days with 100 μM t-BHP (to inducesenescence) were used as a positive control.

DNA Isolation and Quantification

Pellets of cells were flash frozen in liquid nitrogen and thentransferred for further DNA isolation and processing. Genomic DNA wasisolated from the eight treatment group samples using QIAGEN DNeasyBlood & Tissue Kit according to the manufacturer's protocol. Thepurified DNA was quantified using NANODROP 2000 (Therm FisherScientific, Waltham, Mass.). The purity of the DNA was verified bydetermining the A260/A280 ratio for all samples and the ratio wasconsistently ˜1.8.

DNA Hydroxymethylation by Dot Blotting

DNA samples were prepared by diluting total DNA to final amounts of 2μg, 1 μg, 0.5 μg, and 0.25 μg with 0.1 M NaOH. The samples weredenatured at 95° C. for 10 minutes and cooled quickly on an ice bathfollowed by neutralization with ammonium acetate. Loading sample volumesof 400 μl were prepared by adding equal volumes of 0.1 M NaOH and 2 Mammonium acetate to the denatured DNA. The nitrocellulose membrane waspre-wet in distilled water and placed on the microfiltration apparatus(BIO-DOT, Bio-Rad Laboratories, Inc., Hercules, Calif.) according to themanufacturer's recommendations. A vacuum was applied and the screwsre-tightened to hold the apparatus together. The membrane was rehydratedwith 0.1 M NaOH to prepare for sample application. With vacuum off,denatured DNA was added to sample wells, while all other wells werefilled with the same volume of distilled water to obtain homogenousfiltration. The samples were filtered by applying gentle vacuum,followed by an addition of 0.1 M NaOH to each well. The vacuum wasapplied again until wells were empty. The apparatus was disassembled andthe membrane rinsed with 2×SSC. After air-drying, the membrane wasblocked with 5% skimmed milk in PBS for one hour. The membranes werewashed with PBS and incubated with anti-5hmC overnight. The next day,the membrane was washed with PBS and incubated with anti-rabbitsecondary antibody. The blots were washed and developed using enhancedchemiluminescence (SUPERSIGNAL West Femto Maximum Sensitivity Substratekit, Thermo Fisher Scientific, Waltham Mass.) by auto-exposure settingson the CHEMIDOC Touch Imaging System (Bio-Rad Laboratories, Inc.,Hercules, Calif.). Data were quantified by densitometry and analyzedusing Image Lab software by applying background subtraction andapproximated for linearity.

Gene Expression Analysis by Real-Time Quantitative PCR and RNASequencing

Pellets of thawed samples described above were resuspended in QIAZOLlysis agent (Qiagen, Hilden, Germany) for further RNA isolation andprocessing. RNA was isolated using the miRNeasy Mini Kit as per themanufacturer's protocol for cultured cells and cell pellets. Thepurified RNA was quantified using a NANODROP 2000 (Therm FisherScientific, Waltham, Mass.) device to determine concentration.

For qRT-PCR studies, 800 ng of RNA was used for reverse transcription tomake cDNA using SuperScript III First-Strand Synthesis System (ThermoFisher Scientific, Waltham, Mass.). cDNA was diluted to a concentrationof 4 ng/μ.1 and real-time qPCR was performed with 10 ng cDNA per 10 μlreaction with QuantiTect SYBR Green PCR Kit (Qiagen, Hilden, Germany) onCFX384 Real-Time PCR detection system (Bio-Rad Laboratories, Inc.,Hercules, Calif.). The list of genes and their primer sequences areprovided in Table 1. Melt curves were analyzed using the comparative CTmethod (Schmittgen et al., 2008. Nat Protoc 3:1101-1108) with GAPDH asan internal control gene.

High-throughput mRNA sequencing was performed on RNA isolated from theeight sample groups followed by bioinformatics analysis as describedpreviously (Dudakovic et al., 2014. J Cell Biochem 115:1816-1828;Dudakovic et al., 2015. J Biol Chem 290: 27604-27617). Gene expressionis expressed in reads per kilobase pair per million mapped reads (RPKM).Sequencing data are available at National Center for BiotechnologyInformation using Gene Expression Omnibus accession number GSE88946.

Example 6 Sample Preparation

For Raman measurements, experiment solution was made by dissolvingosmolyte into Dulbecco's Phosphate Buffered Saline (DPBS) solution.Single osmolyte solutions were prepared to a final concentration of 600mM. Two multicomponent osmolyte solutions, SSE-A (450 mM sucrose, 60 mMsorbitol, 70 mM ectoine) and SSE-B (450 mM sucrose, 300 sorbitol, 105 mMectoine), also were also prepared.

Lysozyme powder was dissolved into each solution reaching finalconcentration of 100 mg/ml and then refrigerated. Lysozyme in DPBSsolution was used as control. For DSC measurements, experiment solutionwas made by dissolving osmolyte into 20 mM HEPES buffer and theconcentration of osmolyte in each solution remained same as in DPBSsolution. Lysozyme powder was dissolved into each solution reachingfinal concentration of 1 mg/ml and kept refrigerated. Lysozyme in HEPESbuffer was used as control. The pH of solutions was 7.3 measured byBeckman 350 meter.

TABLE 16 Denaturation temperature T_(m) of lysozyme heating in variousosmolyte solution Osmolyte Denaturation temperature T_(m) ° C. SSE-A75.0 SSE-B 78.9 Ectoine 79.6 Sorbitol 83.1 Sucrose 84.4 HEPES 85.1

Confocal Raman System

Confocal Raman Microspectroscopy (CRM) measurements were conducted usinga confocal Raman microscope system (Alpha 300 R, WITec WissenschaftlicheInstrumente und Technologie GmbH, Ulm, Germany) with a spectrometer(UHTS 300, WITec Wissenschaftliche Instrumente und Technologie GmbH,Ulm, Germany) and detector (DV401 CCD, Andor Technology Ltd., Belfast,United Kingdom) with 600/mm grating. The spectrometer was calibratedwith a Mercury-argon lamp. A wavelength of 532 nm Nd:YAG laser poweredat 10 mw was used as an excitation source. The laser was transmitted tothe microscopy in a singer fiber. A 100× air objective (NA 0.90; NikonInstruments, Melville, N.Y.) was used for focusing the 532-nm excitationlaser to the sample. Samples were frozen using a controlled temperaturestage as previously described (Dong et al., 2010. BiophysJ99(8):2453-2459).

Differential Scanning Calorimetry

Differential scanning calorimetry was performed on a differentialscanning calorimeter (Q1000, TA Instruments, New Castle, Del.).

Osmolyte solutions containing lysozyme were heated in order to determinethe denaturation temperature of lysozyme. Experimental solutions wereheated from room temperature to 90° C. at 1° C./min. Specific heat,C_(p), was calculated from the heat flow data assuming

$C_{p} = {\frac{1}{m}{\frac{\delta \; Q}{\Delta \; T}.}}$

C_(p) as a function of temperature were analyzed using OriginProsoftware (OriginLab Corp., Northampton, Mass.) to subtract curvebaseline. The midpoint temperature, T_(m), was obtained as thetemperature corresponding to the maximum specific heat value from thebaseline and the denaturation temperature was assumed to be T_(m) ³².

Osmolyte solutions without lysozyme were also frozen in order tocharacterize phase changes observed in the solutions during freezing.Experimental solutions were frozen to −150° C. using the followingprotocol:

1. Set starting temperature to 20° C.

2. Cool to −150° C. at 10° C./min

3. Hold for 3 min at −150° C.

4. Warm to 20° C. at 10° C./min

The heat release as a function of temperature was used to determine anyphase transition such as glass formation or eutectic formation.

Statistics

Averages plus or minus standard error of the mean are reported unlessotherwise noted. Student's t-tests were performed to determinestatistically significant differences of ice crystal area andellipticity between different osmolyte solutions (P<0.01), as well asα-helix peak intensity of lysozyme frozen in different osmolyte solution(P<0.05) and MSCs recovery in SSE-A and SSE-B solution (P<0.05).

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference in their entirety. In theevent that any inconsistency exists between the disclosure of thepresent application and the disclosure(s) of any document incorporatedherein by reference, the disclosure of the present application shallgovern. The foregoing detailed description and examples have been givenfor clarity of understanding only. No unnecessary limitations are to beunderstood therefrom. The invention is not limited to the exact detailsshown and described, for variations obvious to one skilled in the artwill be included within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

1-18. (canceled)
 19. A method of cryopreserving a cell, the method comprising: adding a cell to a cryopreservative composition comprising: a sugar component; and a sugar alcohol component; wherein total concentration of sugar alcohol components in the composition is between 0.1 M and 2 M, with the proviso that the composition includes less than a cryopreservative amount of dimethyl sulfoxide (DMSO); and freezing the composition.
 20. The method of claim 19, further comprising: storing the frozen composition at a temperature below 0° C.; thawing the composition; removing the cell from the thawed composition; and culturing the cell under conditions effective for the cell to remain viable.
 21. The method of claim 19, wherein the sugar component is provided at a concentration of 0.1 mM to 300 mM.
 22. The method of claim 19, wherein the sugar component is provided at a concentration of 10 mM to 300 mM.
 23. The method of claim 19, wherein the sugar component comprises trehalose, sucrose, lactose, maltose, or a combination thereof.
 24. The method of claim 19, wherein the sugar alcohol component is provided at a concentration of 0.1 M to 1.4 M.
 25. The method of claim 19, wherein the sugar alcohol component is provided at a concentration of 0.1 M to 0.6 M.
 26. The method of claim 19, wherein the sugar alcohol component comprises sorbitol, ethylene glycol, inositol, xylitol, mannitol, or a combination thereof.
 27. The method of claim 19, wherein the sugar alcohol component comprises glycerol at a concentration of 0.6 M to 1.4 M.
 28. The method of claim 19, further comprising an additive component comprising an amino acid.
 29. The method of claim 19, wherein less than a cryopreservative amount of dimethyl sulfoxide (DMSO) is an amount of DMSO that provides no more cryoprotection than the remaining components of the composition without the DMSO.
 30. The method of claim 19, wherein freezing the composition comprises at least one round of cooling, re-warming, and further cooling.
 31. A method of cryopreserving a cell, the method comprising: adding a cell to a cryopreservative composition comprising: a sugar component comprising a disaccharide; and a sugar alcohol component; wherein total concentration of sugar alcohol components in the composition is no more than 2 M, with the proviso that the composition includes less than a cryopreservative amount of dimethyl sulfoxide (DMSO); and freezing the composition.
 32. The method of claim 31, further comprising: storing the frozen composition at a temperature below 0° C.; thawing the composition; removing the cell from the thawed composition; and culturing the cell under conditions effective for the cell to remain viable.
 33. The method of claim 31, wherein the sugar component is provided at a concentration of 0.1 mM to 300 mM.
 34. The method of claim 31, wherein the sugar component comprises trehalose, sucrose, lactose, maltose, or a combination thereof.
 35. The method of claim 31, wherein the sugar alcohol component comprises glycerol, sorbitol, ethylene glycol, inositol, xylitol, mannitol, or a combination thereof.
 36. A method of cryopreserving a cell, the method comprising: adding a cell to a cryopreservative composition comprising: a sugar component comprising a disaccharide, the sugar component being provided at a concentration of up to 300 mM; a sugar alcohol component at a concentration of 0.1 M to 2 M; and less than a cryopreservative amount of dimethyl sulfoxide (DMSO); and freezing the composition.
 37. The method of claim 36, further comprising: storing the frozen composition at a temperature below 0° C.; thawing the composition; removing the cell from the thawed composition; and culturing the cell under conditions effective for the cell to remain viable.
 38. The method of claim 36, wherein the sugar component comprises trehalose, sucrose, lactose, maltose, or a combination thereof, and wherein the sugar alcohol component comprises glycerol, sorbitol, ethylene glycol, inositol, xylitol, mannitol, or a combination thereof. 